Veterinary Parasitology 175 (2011) 2026

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Veterinary Parasitology
journal homepage: www.elsevier.com/locate/vetpar

The potential for zoonotic transmission of Giardia duodenalis and

Cryptosporidium spp. from beef and dairy cattle in Ontario, Canada
Brent Dixon a, , Lorna Parrington a , Angela Cook b , Katarina Pintar b ,
Frank Pollari b , David Kelton c , Jeffrey Farber a
a
b
c

Bureau of Microbial Hazards, Health Canada, 251 Sir Frederick Banting Driveway, P.L. 2204E, Ottawa, Ontario, Canada K1A 0K9
C-EnterNet, Laboratory for Foodborne Zoonoses, Public Health Agency of Canada, 120-255 Woodlawn Road West, Guelph, Ontario, Canada N1H 8J1
Department of Population Medicine, Ontario Veterinary College, University of Guelph, 50 Stone Road, Guelph, Ontario, Canada N1G 2W1

a r t i c l e

i n f o

Article history:
Received 3 June 2010
Received in revised form 1 September 2010
Accepted 29 September 2010
Keywords:
Giardia duodenalis
Cryptosporidium
Dairy cattle
Beef cattle
Genotype
Zoonotic

a b s t r a c t
The objective of this study was to compare the occurrence and the genotypes and species
of Giardia duodenalis and Cryptosporidium spp. in beef and dairy cattle from farms in the
Regional Municipality of Waterloo, Ontario, in an effort to determine the potential for
zoonotic transmission from these animals. Pooled manure samples were collected from 45
dairy cattle farms and 30 beef cattle farms. The presence of Giardia cysts and Cryptosporidium oocysts was determined by immunouorescence microscopy, while nested-PCR and
DNA sequencing were used to determine genotypes and species. The overall farm prevalence was very high for both Giardia and Cryptosporidium, and was similar for dairy cattle
farms (96 and 64%, respectively) and beef cattle farms (97 and 63%, respectively). However,
on dairy cattle farms, G. duodenalis and Cryptosporidium spp. were detected in 44% and 6%
of total pooled pen manure samples, respectively, with the occurrence of both parasites
being generally higher in calves than in older animals. Most Giardia isolates were identied
as either the host-adapted genotype G. duodenalis Assemblage E or the zoonotic Assemblage B. Cryptosporidium parvum and Cryptosporidium andersoni were the most frequently
identied species in dairy cattle, while the non-zoonotic species Cryptosporidium ryanae
and Cryptosporidium bovis were also found. On beef cattle farms, 72% and 27% of the total
pooled pen manure samples were positive for Giardia and Cryptosporidium, respectively,
with no obvious correlation with age. All Giardia isolates in beef cattle were identied as
G. duodenalis Assemblage E, while all Cryptosporidium isolates were identied by sequence
analysis as C. andersoni, although microscopic analyses, and subsequent restriction fragment length polymorphism analyses, indicated that other Cryptosporidium species were
also present. The results of this study indicate that although Giardia and Cryptosporidium
were identied in a higher overall percentage of the pooled beef cattle manure samples
than in dairy cattle, rmly established zoonotic genotypes and species were much more
common in dairy cattle than in beef cattle in this region. Dairy cattle, and especially dairy
calves, may, therefore, pose a greater risk of infection to humans than beef cattle. However, these results may also provide evidence of potential zooanthroponotic transmission
(human to animal).
Crown Copyright 2010 Published by Elsevier B.V. All rights reserved.

1. Introduction
Corresponding author at: Microbiology Research Division, Banting
Research Centre, 251 Sir Frederick Banting Driveway, P.L. 2204E, Ottawa,
Ontario, Canada K1A 0K9. Tel.: +1 613 957 0904; fax: +1 613 941 0280.
E-mail address: Brent.Dixon@hc-sc.gc.ca (B. Dixon).

Giardia duodenalis and Cryptosporidium spp. are common protozoan parasites responsible for enteric illness
in humans and animals worldwide. Human transmis-

0304-4017/$ see front matter. Crown Copyright 2010 Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.vetpar.2010.09.032

B. Dixon et al. / Veterinary Parasitology 175 (2011) 2026

sion is predominantly through the fecaloral route

(person-to-person) and contaminated water (drinking and
recreational). However, there has also been considerable interest and discussion surrounding the potential
for zoonotic transmission of these pathogens, particularly from livestock (Thompson, 2004; Olson et al., 2004;
Hunter and Thompson, 2005; OHandley and Olson, 2006;
OHandley, 2007; Xiao and Feng, 2008; Xiao and Fayer,
2008). Transmission of this type may occur through either
direct contact in the case of farmers, veterinarians, and petting zoos, or through indirect routes such as contaminated
surface water or foods (Dixon, 2009).
A high prevalence of both Giardia and Cryptosporidium
has been reported worldwide in dairy and beef cattle. The
majority of these studies, however, have involved dairy
calves, in which the prevalence of both parasites has generally been reported to be very high, with a number of studies
reporting a 100% cumulative prevalence of both Giardia
and Cryptosporidium (OHandley et al., 1999; Santn et al.,
2008, 2009; Coklin et al., 2010). Studies in Canada have
reported prevalences in dairy calves of between 45.1 and
73% for G. duodenalis, and between 6.2 and 40.6% for Cryptosporidium spp. (Olson et al., 1997a; OHandley et al., 2000;
Trotz-Williams et al., 2005; Coklin et al., 2007, 2009). In
the U.S., Trout et al. (2004, 2005) examined the prevalence
and genotypes of G. duodenalis in pre- and post-weaned
dairy calves from several states. These authors reported a
widely varying prevalence of 993%, with an average of 40%
in pre-weaned calves and an overall prevalence of 52% in
post-weaned calves. Cryptosporidium spp. prevalences of
between 7.5% and 49% have been reported in dairy calves
in the U.S. (Garber et al., 1994; Santn et al., 2004; Nydam
et al., 2005; Starkey et al., 2005; Fayer et al., 2010). Several studies have examined Cryptosporidium and Giardia
infections in adult dairy cattle in North America, and have
reported a generally lower prevalence than in calves. In
Canada, Giardia prevalence in cows was reported as 28.3%
(Coklin et al., 2007) and 49% (Uehlinger et al., 2006). Both
studies reported a complete absence of Cryptosporidium
spp. in these animals. In the U.S., the prevalence of Giardia
in dairy cows ranged from 0 to 64%, while Cryptosporidium
ranged from 5.7 to 12.5% (Fayer et al., 2000, 2007; Trout et
al., 2007).
In beef calves, Ralston et al. (2003) reported a 100%
cumulative prevalence of Giardia in Alberta, Canada, while
point prevalence studies in North America have demonstrated Giardia infection rates of between 22.6 and 37.3%
(Olson et al., 1997b; Fayer et al., 2000; Appelbee et al., 2003;
McAllister et al., 2005; Gow and Waldner, 2006). Giardia
prevalence rates of 8.7 to 17% have been reported in adult
beef cattle (Olson et al., 1997b; McAllister et al., 2005; Gow
and Waldner, 2006). Ralston et al. (2003) reported a cumulative Cryptosporidium prevalence of only 5% in beef calves
in Alberta, Canada. Point prevalence studies have similarly
shown a relatively low Cryptosporidium prevalence in beef
calves of between 3.1 and 15% in Canada (Olson et al.,
1997b; McAllister et al., 2005; Gow and Waldner, 2006),
and between 0 and 13% in California (Atwill et al., 1999).
One study, however, demonstrated a relatively high Cryptosporidium prevalence of 28.8% in 79-month-old beef
calves (Fayer et al., 2000). The prevalence of Cryptosporid-

21

ium in adult beef cattle has been reported to be between 0.6

and 28.9% in North America (Olson et al., 1997b; Atwill et
al., 1999, 2003; McAllister et al., 2005; Gow and Waldner,
2006).
Three different molecular assemblages of G. duodenalis
have been reported in cattle. Assemblage A, which has a
wide host range including humans, and the host adapted
hoofed livestock Assemblage E, are frequently recognized
in cattle. Assemblage B, which represents the second G.
duodenalis genotype found in humans, also has a wide host
range, but has only more recently been identied in cattle (van Keulen et al., 2002; Lalle et al., 2005; Mendonca
et al., 2007; Coklin et al., 2007; Winkworth et al., 2008).
Numerous studies have reported a higher prevalence of
Assemblage E in cattle than Assemblage A (OHandley et
al., 2000; van Keulen et al., 2002; Appelbee et al., 2003;
Trout et al., 2004, 2005, 2006, 2007; OHandley and Olson,
2006), although Uehlinger et al. (2006) reported comparable prevalence rates for both Assemblages in adult dairy
cattle, suggesting a greater risk of zoonotic transmission
than previously thought. Geurden et al. (2008) reported a
higher prevalence of Assemblage A than E in dairy calves in
Belgium, while Assemblage E predominated in beef calves.
Similarly, Santn et al. (2009) demonstrated that infections
with the zoonotic Assemblage A are more common in dairy
calves than previously reported, suggesting that calves may
be an important source of human infection.
Cattle have been reported as primary hosts for four
Cryptosporidium species (Fayer et al., 2008). Cryptosporidium parvum has a very wide host range and is thought
to be the most important in terms of zoonotic transmission, as it is frequently identied in human cases. Of the
three other species found in cattle, Cryptosporidium bovis
and Cryptosporidium ryanae (previously known as the deerlike genotype) are host specic and non-zoonotic, while
Cryptosporidium andersoni has been reported in only four
human cases (Leoni et al., 2006; Morse et al., 2007). The
species of Cryptosporidium found in dairy cattle are agespecic with C. parvum highly prevalent in pre-weaned
calves and being replaced with C. bovis, C. ryanae and C.
andersoni in post-weaned calves and older animals (Santn
et al., 2004, 2008; Fayer et al., 2006, 2007; Thompson et al.,
2007; Keshavarz et al., 2009).
The majority of prevalence and molecular characterization studies of G. duodenalis and Cryptosporidium spp. in
cattle have involved dairy cattle, and particularly calves.
Less data are available with regard to beef cattle, and only
a few studies have directly compared the prevalence, or the
genotypes and species of G. duodenalis and Cryptosporidium
spp. in both dairy and beef cattle from the same region. For
example, Geurden et al. (2008) reported a higher prevalence of Giardia infection in beef calves than in dairy calves
in Belgium, and Kvc et al. (2006) looked at sources of transmission of Cryptosporidium in both dairy calves and beef
calves in the Czech Republic. The objective of this study
was to determine the occurrence, as well as the genotypes
and species, of G. duodenalis and Cryptosporidium spp. in
both dairy and beef cattle from the Regional Municipality
of Waterloo, Ontario, in order to determine their relative
risks with regards to the zoonotic transmission of these
pathogens. This work is part of on-going surveillance by the

22

B. Dixon et al. / Veterinary Parasitology 175 (2011) 2026

C-EnterNet Program of the Public Health Agency of Canada

that monitors human enteric illness and enteric pathogen
exposure through food animals, retail foods, and water at
Canadian sentinel sites.

Eclipse E600 epiuorescence microscope (Nikon Canada

Inc. Instruments, Mississauga, Ontario). Wherever possible,
Giardia cysts and Cryptosporidium oocysts were conrmed
using differential interference contrast (DIC) microscopy.

2. Materials and methods

2.4. DNA extraction

2.1. Sample collection

Total DNA was extracted from each sucrose otation

concentrated sample using the DNeasy Tissue Kit (Qiagen
Inc., Mississauga, ON), using a slightly modied protocol.
A total of 200 l of sucrose otation concentrated sample was added to 1.5 ml microcentrifuge tubes and lysed
overnight at 56 C using 180 l of lysis buffer and 20 l
of proteinase K (20 mg/ml) supplied with the DNeasy Tissue Kit. The subsequent lysate was transferred to a new
tube, and genomic DNA was isolated using the DNeasy
Tissue Kit following manufacturers instructions. To purify
the DNA, the nucleic acid was eluted in 150 l of elution
buffer.

A total of 179 pooled manure samples were collected

from 45 dairy cattle farms in the Regional Municipality of
Waterloo, Ontario, from May to October, 2006. At each visit,
three fresh pooled manure samples (n = 138), from different age groups or different areas of the farm, as well as one
stored manure sample (n = 41), were collected. Similarly,
112 pooled manure samples were collected from 30 beef
cattle farms in the same region between February 2007 and
March 2008, with a total of 87 fresh pen manure samples
and 25 stored manure samples. All manure samples were
collected in sterile pre-labeled plastic cups, which were
then capped and immediately placed into insulated containers packed with ice or cold packs. The samples were
shipped to Ottawa, Ontario, by overnight courier where
they were refrigerated and processed within one to three
days of collection.
2.2. Sample preparation
The sucrose otation technique of Uehlinger et al.
(2006) was used with the following modication. To 20 g
of feces, 35 ml of phosphate buffered saline with 0.01%
Triton X-100 (PBS-TX) pH 7.4 was added, and the slurry
was thoroughly mixed with an applicator. The suspension was then passed through four layers of gauze. Sample
preparation for the beef manure samples was further modied at this point as follows; ltered suspension was
divided into equal volumes, and layered over 15 ml of
1 M sucrose solution (specic gravity 1.13) in two conical
50 ml Falcon tubes. Samples were centrifuged at 800 g
for 5 min. Following centrifugation, the interface and the
upper layer of liquid from each tube was transferred to a
clean tube, the volume was adjusted to 45 ml with PBS-TX
and re-centrifuged at 1500 g for 10 min. The supernatant
was decanted and the pellet was re-suspended in 1 ml
PBS and used for microscopy and molecular characterization.
2.3. Microscopy
For each sample, 200 l of suspension was transferred to
a microcentrifuge tube. Fluorescein isothiocyanate (FITC)
labeled monoclonal antibody solution (50 l each of Giardia-Glo and Crypt-a-Glo, Waterborne Inc., New Orleans,
Louisiana) was added to the tube, which was then vortexed. The tube was then incubated at room temperature
for 45 min in the dark. After incubation, the excess antibody was washed by adding 1 ml of PBS, vortexing, and
centrifuging at 10,000 g for 10 min. The supernatant was
then pipetted off, leaving 100 l which was then used to
re-suspend the pellet. Twenty l was added to a microscope slide, coverslipped, and examined at 200 on a Nikon

2.5. Giardia PCR

Nested-PCR was performed to amplify fragments of the
16S rRNA gene and the -giardin gene for Giardia. Amplication of fragments of the 16S rRNA gene was performed
as described in Coklin et al. (2007). Amplication of fragments of the -giardin gene was performed as described in
Cacci et al. (2002). PCR products were separated by electrophoresis on 1.2% (w/v) agarose gels, and visualized by
ethidium bromide staining.
2.6. Cryptosporidium PCR
Nested-PCR was performed to amplify fragments of the
18S rRNA gene and the heat shock protein-70 (HSP-70)
gene for Cryptosporidium. Amplication of fragments of the
18S rRNA gene was performed as described in Coklin et
al. (2010). PCR products were separated by electrophoresis
on 1.2% (w/v) agarose gels, and visualized by ethidium bromide staining. A restriction fragment length polymorphism
(RFLP) analysis of 18S rRNA-PCR amplicons for the identication of Cryptosporidium species was performed using
the restriction enzymes SspI, VspI, and MboII (Xiao et al.,
1999; Feng et al., 2007). RFLP amplication of fragments of
the HSP-70 gene was performed as described in Coklin et
al. (2007). RFLP banding patterns were observed following
electrophoresis on 2% (w/v) agarose gels.
2.7. DNA sequence analysis
DNA sequencing was performed at the McGill University
and Genome Quebec Innovation Centre, Montreal, Quebec,
using a 3730xl DNA Analyser (Applied Biosystems, Foster
City, California). PCR products were puried with Millipore MultiScreen HTS PCR 96-well plates, and sequenced in
both directions using the same PCR primers as for the original amplications. DNA sequences were aligned using the
Genetic Computer Group sequence analysis package (version 10.3, Madison, Wisconsin) or Bioedit (version 7.0.9,
Hall, 1999).

B. Dixon et al. / Veterinary Parasitology 175 (2011) 2026

23

Table 1
G. duodenalis and Cryptosporidium spp. in pooled dairy cattle manure.
Sample type (n)

Calves (11)
Heifers (48)
Cows (79)
Stored manure (41)
Total pens (138)

Microscopy

PCR

Giardia positive (%)

Cryptosporidium positive (%)

Giardia positive (%)

Cryptosporidium positive (%)

7 (64)
31 (65)
23 (29)
11 (27)
61 (44)

4 (36)
3 (6)
1 (1)
6 (15)
8 (6)

10 (91)
28 (58)
31 (39)
19 (46)
69 (50)

5 (46)
9 (19)
10 (13)
15 (37)
24 (17)

Table 2
G. duodenalis and Cryptosporidium spp. in pooled beef cattle manure.
Sample type (n)

Calves (10)
Heifers (18)
Steers (47)
Cows (4)
Bulls (1)
Cow/calf (5)
Stored manure (25)
Total pens (85)

Microscopy

PCR

Giardia positive (%)

Cryptosporidium positive (%)

Giardia positive (%)

Cryptosporidium positive (%)

7 (70)
13 (72)
38 (81)
0
1 (100)
2 (40)
10 (40)
61 (72)

1 (10)
8 (44)
14 (30)
0
0
0
4 (16)
23 (27)

7 (70)
12 (67)
42 (89)
2 (50)
1 (100)
1 (20)
10 (40)
65 (77)

0
8 (44)
16 (34)
0
0
0
7 (28)
24 (28)

3. Results
Of the 45 dairy cattle farms sampled in this study, Giardia cysts were detected on 43 (96%), and Cryptosporidium
oocysts were detected on 29 (64%). Only two farms were
found to be negative for both parasites. Of the 30 beef cattle farms, Giardia cysts were detected on 29 (97%), and
Cryptosporidium oocysts were detected on 19 (63%), and
only one farm was found to be negative for both parasites.
Farm prevalence was based on the total number of positive
pen manure and stored manure samples detected by either
microscopy or PCR.
The overall occurrence of G. duodenalis cysts and Cryptosporidium spp. oocysts in the 138 pooled pen manure
samples tested from the dairy farms, determined by
microscopy, was 44% and 6%, respectively. The occurrence
of G. duodenalis cysts and Cryptosporidium spp. oocysts
determined by PCR was 50% and 17% of the samples, respectively. When data were analysed according to the age of
the animals, the occurrence of both parasites was generally highest in calves, followed by heifers and then adult
cows (Table 1). Of the 85 pooled pen manure samples tested
from the beef cattle farms, 72% and 27% were positive
for G. duodenalis and Cryptosporidium spp., respectively,
by microscopy, and 77% and 28%, respectively, by PCR
(Table 2). While the total pen prevalence for both Giardia
and Cryptosporidium was much higher in beef cattle than
in dairy cattle, a considerably smaller proportion of the
pooled manure samples from beef calves were positive for

Cryptosporidium than the pooled manure from dairy calves.

Other than this lower occurrence of Cryptosporidium in beef
calves, there was no obvious correlation between age of
the beef cattle and the occurrence of either parasite, as was
observed with the dairy cattle, possibly due to the greater
number of age categories of beef cattle, and the smaller
number of samples in each.
DNA sequence data were available for 35 Giardia positive pooled pen manure samples and 15 Cryptosporidium
positive pooled pen manure samples from the dairy cattle. Most Giardia isolates were identied as either the
host-adapted hoofed livestock genotype G. duodenalis
Assemblage E (57%) or the zoonotic Assemblage B (34%),
while zoonotic Assemblage A was less common (9%).
Notably, Assemblage B was the most common genotype
in calves, while Assemblage E was the most common in
both heifers and cows (Table 3). The zoonotic species C.
parvum (50%), as well as C. andersoni (31%), were the most
frequently identied species in dairy cattle, with C. parvum
predominating in calves, and C. andersoni only present in
heifers and cows. The non-zoonotic species C. ryanae and C.
bovis (13% and 6%, respectively), were present in younger
animals only (Table 4).
Based on DNA sequencing of 63 Giardia positive pooled
pen manure samples and 17 Cryptosporidium positive
pooled pen manure samples, all G. duodenalis isolates in
beef cattle were identied as Assemblage E (Table 5),
while all Cryptosporidium spp. isolates were identied by
sequence analysis as C. andersoni (Table 6). Microscopical

Table 3
Molecular characterization of G. duodenalis in dairy cattle.
Sample type (number sequenced)

Assemblage E number (%)

Assemblage B number (%)

Assemblage A number (%)

Calves (8)
Heifers (8)
Cows (19)
Total (35)

3 (38)
5 (63)
12 (63)
20 (57)

4 (50)
3 (38)
5 (26)
12 (34)

1 (13)
0
2 (11)
3 (9)

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B. Dixon et al. / Veterinary Parasitology 175 (2011) 2026

Table 4
Molecular characterization of Cryptosporidium spp. in dairy cattle.
Sample type (number sequenced)

C. parvum number (%)

C. andersoni number (%)

C. ryanae number (%)

C. bovis number (%)

Calves (5)
Heifers (4)
Cows (6)
Total (15)

4 (80)
1 (25)
3 (50)
8 (50)

0
2 (50)
3 (50)
5 (31)

1 (20)
1 (25)
0
2 (13)

0
1 (25)a
0
1 (6)

One pooled heifer sample showed both C. bovis by HSP-70 and C. ryanae by 18S.

Table 5
Molecular characterization of G. duodenalis in beef cattle.
Sample type (number sequenced)

Assemblage E number (%)

Assemblage B number (%)

Assemblage A number (%)

Calves (7)
Heifers (11)
Steers (41)
Cows (2)
Bulls (1)
Cow/calf (1)
Total (63)

7 (100)
11 (100)
41 (100)
2 (100)
1 (100)
1 (100)
63 (100)

0
0
0
0
0
0
0

0
0
0
0
0
0
0

Table 6
Molecular characterization of Cryptosporidium spp. in beef cattle.
Sample typea (number sequenced)

C. parvum number (%)

C. andersoni number (%)

C. ryanae number (%)

C. bovis number (%)

Calves (1)
Heifers (5)
Steers (11)
Total (17)

0
0
0b
0

1 (100)
5 (100)
11 (100)
17 (100)

0
0
0
0

0
0
0c
0

a
b
c

There were no Cryptosporidium positives in samples from cows, bulls or cow/calves.

C. parvum was identied in 3 steers by PCR-RFLP.
C. bovis was identied in 1 steer by PCR-RFLP.

analysis, however, revealed the presence of species other

than C. andersoni in some of these samples based on their
smaller oocyst size. Subsequent PCR-RFLP analysis on the
18S rRNA-PCR amplicons of eight such samples indicated
that, in addition to C. andersoni, C. parvum was present in
three, and C. bovis was present in one (all steers).
PCR based on amplication of a portion of the small subunit (16S and 18S) rRNA genes was more sensitive than the
-giardin gene or the HSP-70 gene for detecting G. duodenalis and Cryptosporidium spp., respectively, in both dairy
and beef cattle manure. However, in both dairy and beef
cattle manure, PCR based on amplication of the -giardin
gene identied a small number of G. duodenalis positives
that the 16S rRNA gene did not, justifying the use of more
than one gene in the PCR-based detection of these parasites.
4. Discussion
The present study represents one of very few directly
comparing the occurrence and the zoonotic potential of
G. duodenalis and Cryptosporidium spp. in dairy and beef
cattle in the same region. The overall farm prevalence
of G. duodenalis was very high in this study, with both
dairy cattle and beef cattle farms approaching 100%. Cryptosporidium spp. were less prevalent than G. duodenalis,
but were still present on the majority of both types of
farms. High farm prevalences have similarly been reported
in a number of other studies (Olson et al., 1997a,b; Ruest
et al., 1998; Appelbee et al., 2003; Trotz-Williams et al.,
2005; McAllister et al., 2005; Kvc et al., 2006; Gow and

Waldner, 2006; Trout et al., 2007; Fayer et al., 2007; Coklin

et al., 2009). Based on total pen results, and without taking age into account, the occurrence of G. duodenalis and
Cryptosporidium spp. was considerably higher in the pooled
pen manure samples collected from beef cattle farms than
those from dairy cattle farms, although this was due largely
to the higher occurrence of these parasites in older beef
cattle. A higher prevalence of G. duodenalis in beef calves
than in dairy calves has only rarely been reported previously (Geurden et al., 2008). The higher occurrence of
Cryptosporidium spp. in dairy calves than in beef calves
in the present study is in agreement with other studies
(OHandley and Olson, 2006; Kvc et al., 2006). A variety
of reasons have been put forth to explain this generally
higher prevalence of Giardia and Cryptosporidium in dairy
calves (Atwill et al., 1999; Olson et al., 2004; McAllister et
al., 2005; OHandley, 2007).
Zoonotic genotypes and species of G. duodenalis and
Cryptosporidium spp. were much more common in dairy
cattle than in beef cattle in this region. Dairy calves, in
particular, are of concern as the zoonotic Assemblage B
was the most common genotype of G. duodenalis, and the
zoonotic species, C. parvum, predominated. In contrast, all
G. duodenalis isolates in beef cattle were the non-zoonotic
Assemblage E. Appelbee et al. (2003) similarly reported a
predominance of Assemblage E in beef calves in Alberta,
and concluded that these animals pose a minimal zoonotic
threat. Similarly, all Cryptosporidium spp. isolates from
beef cattle manure in the present study were identied
as non-zoonotic. The presence of other species, includ-

B. Dixon et al. / Veterinary Parasitology 175 (2011) 2026

ing C. parvum, detected in a small number of samples

by microscopy and PCR-RFLP analysis may have been the
result of the preferential PCR amplication of the predominant species, C. andersoni, as has been recently reported
in cattle in India (Paul et al., 2009). Preferential amplication and mixed infections have been discussed by Santn
et al. (2009). The results of this study support the conclusion of OHandley (2007) that beef calves may pose less
of a risk of zoonotic transmission than dairy calves. While
not commonly associated with human infections, C. andersoni has been reported in a small number of cases (Leoni
et al., 2006; Morse et al., 2007), suggesting that it may also
pose some zoonotic risk. This is of some concern in this
particular region of Ontario, as studies have indicated that
C. andersoni is, in fact, the predominant species detected in
surface waters (Government of Canada, C-EnterNet Annual
Reports, 20062008). This nding corresponds to the high
prevalence of C. andersoni in both dairy and beef cattle in
the present study, and provides further evidence for cattle
as an important source of contamination of surface water
sources. However, in a limited number of samples from
these same surface waters, only Giardia microti have been
identied to date.
The occurrence of G. duodenalis and Cryptosporidium
spp. in pooled dairy cattle manure samples, particularly
those from young animals, along with the high prevalence
of zoonotic genotypes and species, suggests that there may
be a risk of transmission to farmers, veterinarians, and farm
or petting zoo visitors by means of direct contact, and to the
general human population in the region through the cyst
or oocyst contamination of surface water (through agricultural runoff) or produce (through manure application
to crop lands, or irrigation and processing with contaminated water). However, the presence of similar genotypes
and species in cattle and humans is not necessarily evidence for transmission from cattle to humans, and direct
evidence for zoonotic transmission from cattle and other
livestock remains rather scant (Dixon, 2009). These similar
genotypes and species may also be indicative of zooanthroponotic transmission (human to animal). While there is
considerable evidence for transmission of Giardia and Cryptosporidium from cow-to-calf or from calf-to-calf (Garber
et al., 1994; OHandley et al., 1999; Faubert and Litvinsky,
2000; Fayer et al., 2000; Huetink et al., 2001; Ralston et
al., 2003; Becher et al., 2004; Gow and Waldner, 2006), a
high prevalence of zoonotic G. duodenalis Assemblages, and
C. parvum, in cattle is also suggestive of a human source
of infection in these animals (OHandley and Olson, 2006;
Coklin et al., 2007; Geurden et al., 2008), whereby cattle may be exposed to cysts and oocysts of human origin
through sewage contaminated drinking water or feed, or
through direct contact with infected animal handlers. As
evidence of this, in the present study, beef cattle were
only infected with non-zoonotic genotypes and species,
whereas dairy cattle, which generally have much more
human contact, also had zoonotic isolates. It is not clear,
however, if these results are evidence for on-going zooanthroponotic transmission or whether, once established,
these genotypes and species have continued to circulate
among these animals. Prevalence testing and molecular
characterization, including subtyping of Giardia and Cryp-

25

tosporidium in fecal samples from veterinarians, farmers

and other animal handlers would be benecial in providing better insight on the possible transmission dynamics of
these parasites.
The results of this study indicate that both G. duodenalis and Cryptosporidium spp. are very common on dairy
and beef cattle farms in Ontario. Although both parasites
were identied in a higher percentage of the total pooled
beef cattle manure samples than in dairy cattle, zoonotic
genotypes and species were much more common in dairy
cattle than in beef cattle. Dairy cattle, and especially calves,
may, therefore, pose a greater risk of infection to humans
than beef cattle in this region. However, these results may
also provide evidence of potential zooanthroponotic transmission, and further work on the transmission patterns of
Giardia and Cryptosporidium in cattle is warranted.
Acknowledgements
The authors wish to thank Tatjana Coklin, Themba
Mtimkulu and Oksana Mykytczuk, Bureau of Microbial
Hazards, Health Canada, for technical assistance. Our
thanks also go to Mollie Campbell, University of Guelph,
for her verication of the sample collection spreadsheets,
and to eld technicians from the University of Guelph, for
sample collection. The C-EnterNet program was funded by
the Agriculture Policy Framework initiative of Agriculture
and Agri-Food Canada.
References
Appelbee, A.J., Frederick, L.M., Heitman, T.L., Olson, M.E., 2003. Prevalence and genotyping of Giardia duodenalis from beef calves in Alberta,
Canada. Vet. Parasitol. 112, 289294.
Atwill, E.R., Johnson, E., Klingborg, D.J., Veserat, G.M., Markegard, G.,
Jensen, W.A., Pratt, D.W., Delmas, R.E., George, H.A., Forero, L.C.,
Philips, R.L., Barry, S.J., McDougald, N.K., Gildersleeve, R.R., Frost, W.E.,
1999. Age, geographic, and temporal distribution of fecal shedding of
Cryptosporidium parvum oocysts in cow-calf herds. Am. J. Vet. Res. 60,
420425.
Atwill, E.R., Hoar, B., Pereira, M.D.G.C., Tate, K.W., Rulofson, F., Nader, G.,
2003. Improved quantitative estimates of low environmental loading
and sporadic periparturient shedding of Cryptosporidium parvum in
adult beef cattle. Appl. Environ. Microbiol. 69, 46044610.
Becher, K.A., Robertson, I.D., Fraser, D.M., Palmer, D.G., Thompson, R.C.A.,
2004. Molecular epidemiology of Giardia and Cryptosporidium infections in dairy calves originating from three sources in Western
Australia. Vet. Parasitol. 123, 19.
Cacci, S.M., De Giacomo, M., Pozio, E., 2002. Sequence analysis of the
-giardin gene and development of a polymerase chain reactionrestriction fragment length polymorphism assay to genotype Giardia
duodenalis cysts from human faecal samples. Int. J. Parasitol. 32,
10231030.
Coklin, T., Farber, J., Parrington, L., Dixon, B., 2007. Prevalence and molecular characterization of Giardia duodenalis and Cryptosporidium spp.
in dairy cattle in Ontario, Canada. Vet. Parasitol. 150, 297305.
Coklin, T., Uehlinger, F.D., Farber, J.M., Barkema, H.W., OHandley, R.M.,
Dixon, B.R., 2009. Prevalence and molecular characterization of Cryptosporidium spp. in dairy calves from 11 farms in Prince Edward
Island, Canada. Vet. Parasitol. 160, 323326.
Coklin, T., Farber, J.M., Parrington, L.J., Coklin, Z., Ross, W.H., Dixon, B.R.,
2010. Temporal changes in the prevalence and shedding patterns of
Giardia duodenalis cysts and Cryptosporidium spp. oocysts in dairy
calves at an agricultural college in Ontario, Canada. Can. Vet. J. 51,
841846.
Dixon, B.R., 2009. The role of livestock in the foodborne transmission of
Giardia duodenalis and Cryptosporidium spp. to humans. In: OrtegaPierres, M.G., Cacci, S., Fayer, R., Mank, T., Smith, H., Thompson, R.C.A.
(Eds.), Giardia and Cryptosporidium: From Molecules to Disease. CAB
International, Wallingford, UK, pp. 107122.

26

B. Dixon et al. / Veterinary Parasitology 175 (2011) 2026

Faubert, G.M., Litvinsky, Y., 2000. Natural transmission of Cryptosporidium

parvum between dams and calves on a dairy farm. J. Parasitol. 86,
495500.
Fayer, R., Trout, J.M., Graczyk, T.K., Lewis, E.J., 2000. Prevalence of Cryptosporidium, Giardia and Eimeria infections in post-weaned and adult
cattle on three Maryland farms. Vet. Parasitol. 93, 103112.
Fayer, R., Santn, M., Trout, J.M., Greiner, E., 2006. Prevalence of species and
genotypes of Cryptosporidium found in 1 to 2-year-old dairy cattle in
the eastern United States. Vet. Parasitol. 135, 105112.
Fayer, R., Santn, M., Trout, J.M., 2007. Prevalence of Cryptosporidium
species and genotypes in mature dairy cattle on farms in eastern
United States compared with younger cattle from the same locations.
Vet. Parasitol. 145, 260266.
Fayer, R., Santn, M., Trout, J.M., 2008. Cryptosporidium ryanae n. sp. (Apicomplexa: Cryptosporidiidae) in cattle (Bos taurus). Vet. Parasitol.
156, 191198.
Fayer, R., Santn, M., Dargatz, D., 2010. Species of Cryptosporidium detected
in weaned cattle on cow-calf operations in the United States. Vet.
Parasitol. 170, 187192.
Feng, Y., Ortega, Y., He, G., Das, P., Xu, M., Zhang, X., Fayer, R., Gatei, W.,
Cama, V., Xiao, L., 2007. Wide geographic distribution of Cryptosporidium bovis and the deer-like genotype in bovines. Vet. Parasitol. 144,
19.
Garber, L.P., Salman, M.D., Hurd, H.S., Keefe, T., Schlater, J.L., 1994. Potential
risk factors for Cryptosporidium infection in dairy calves. J. Am. Vet.
Med. Assoc. 205, 8691.
Geurden, T., Geldhof, P., Levecke, B., Martens, C., Berkvens, D., Casaert, S.,
Vercruysse, J., Claerebout, E., 2008. Mixed Giardia duodenalis assemblage A and E infections in calves. Int. J. Parasitol. 38, 259264.
Government of Canada Canadian National Integrated Enteric Pathogen
Surveillance System (C-EnterNet) Annual Reports (20062008).
Guelph, ON: Public Health Agency of Canada. http://www.phacaspc.gc.ca/c-enternet/publications-eng.php.
Gow, S., Waldner, C., 2006. An examination of the prevalence of and risk
factors for shedding of Cryptosporidium spp. and Giardia spp. in cows
and calves from western Canadian cow-calf herds. Vet. Parasitol. 137,
5061.
Hall, T.A., 1999. BioEdit: a user-friendly biological sequence alignment
editor and analysis program for Windows 95/98/NT. Nucl. Acids Symp.
Ser. 41, 9598, http://www.mbio.ncsu.edu/BioEdit/BioEdit.html.
Huetink, R.E.C., van der Giessen, J.W.B., Noordhuizen, J.P.T.M., Ploeger,
H.W., 2001. Epidemiology of Cryptosporidium spp. and Giardia duodenalis on a dairy farm. Vet. Parasitol. 102, 5367.
Hunter, P.R., Thompson, R.C.A., 2005. The zoonotic transmission of Giardia
and Cryptosporidium. Int. J. Parasitol. 35, 11811190.
Keshavarz, A., Haghighi, A., Athari, A., Kazemi, B., Abadi, A., Mojarad,
E.N., 2009. Prevalence and molecular characterization of bovine Cryptosporidium in Qazvin province. Iran. Vet. Parasitol. 160, 316318.
Kvc, M., Kouba, M., Vtovec, J., 2006. Age-related and housing dependence
of Cryptosporidium infection of calves from dairy and beef herds in
South Bohemia, Czech Republic. Vet. Parasitol. 137, 202209.
Lalle, M., Pozio, E., Capelli, G., Bruschi, F., Crotti, D., Caccio, S.M., 2005.
Genetic heterogeneity at the -giardin locus among human and animal isolates of Giardia duodenalis and identication of potentially
zoonotic subgenotypes. Int. J. Parasitol. 35, 207213.
Leoni, F., Amar, C., Nichols, G., Pedraza-Daz, S., McLauchlin, J., 2006.
Genetic analysis of Cryptosporidium from 2414 humans with diarrhea
in England between 1985 and 2000. J. Med. Microbiol. 55, 703707.
McAllister, T.A., Olson, M.E., Fletch, A., Wetzstein, M., Entz, T., 2005. Prevalence of Giardia and Cryptosporidium in beef cows in southern Ontario
and in beef calves in southern British Columbia. Can. Vet. J. 46, 4755.
Mendonca, C., Almeida, A., Castro, A., de Lurdes Delgado, M., Soares,
S., da Costa, J.M.C., Canada, N., 2007. Molecular characterization of
Cryptosporidium and Giardia isolates from cattle from Portugal. Vet.
Parasitol. 147, 4750.
Morse, T.D., Nichols, R.A.B., Grimason, A.M., Campbell, B.M., Tembo, K.C.,
Smith, H.V., 2007. Incidence of cryptosporidiosis species in paediatric
patients in Malawi. Epidemiol. Infect. 135, 13071315.
Nydam, D.V., Lindergard, G., Santucci, F., Schaaf, S.L., Wade, S.E.,
Mohammed, H.O., 2005. Risk of infection with Cryptosporidium
parvum and Cryptosporidium hominis in dairy cattle in the New York
City watershed. Am. J. Vet. Res. 66, 413417.
OHandley, R.M., Olson, M.E., 2006. Giardiasis and cryptosporidiosis in
ruminants. Vet. Clin.: Food Anim. Pract. 22, 623643.
OHandley, R.M., Cockwill, C., McAllister, T.A., Jelinski, M., Morck, D.W.,
Olson, M.E., 1999. Duration of naturally acquired giardiosis and cryptosporidiosis in dairy calves and their association with diarrhea. J. Am.
Vet. Med. Assoc. 214, 391396.

OHandley, R.M., Olson, M.E., Fraser, D., Adams, P., Thompson, R.C.,
2000. Prevalence and genotypic characterization of Giardia in dairy
calves from Western Australia and western Canada. Vet. Parasitol. 90,
193200.
OHandley, R.M., 2007. Cryptosporidium parvum infections in cattle: are
current perceptions accurate? Trends Parasitol. 23, 477480.
Olson, M.E., Guselle, N.J., OHandley, R.M., Swift, M.L., McAllister, T.A., Jelinski, M.D., Morck, D.W., 1997a. Giardia and Cryptosporidium in dairy
calves in British Columbia. Can. Vet. J. 38, 703706.
Olson, M.E., Thorlakson, C.L., Deselliers, L., Morck, D.W., McAllister, T.A.,
1997b. Giardia and Cryptosporidium in Canadian farm animals. Vet.
Parasitol. 68, 375381.
Olson, M.E., OHandley, R.M., Ralston, B.J., McAllister, T.A., Thompson,
R.C.A., 2004. Update on Cryptosporidium and Giardia infections in cattle. Trends Parasitol. 20, 185191.
Paul, S., Chandra, D., Tewari, A.K., Banerjee, P.S., Ray, D.D., Raina, O.K.,
Rao, J.R., 2009. Prevalence of Cryptosporidium andersoni: a molecular epidemiological survey among cattle in India. Vet. Parasitol. 161,
3135.
Ralston, B.J., McAllister, T.A., Olson, M.E., 2003. Prevalence and infection
pattern of naturally acquired giardiasis and cryptosporidiosis in range
beef calves and their dams. Vet. Parasitol. 114, 113122.
Ruest, N., Faubert, G.M., Couture, Y., 1998. Prevalence and geographical
distribution of Giardia spp. and Cryptosporidium spp. in dairy farms in
Quebec. Can. Vet. J. 36, 697700.
Santn, M., Trout, J.M., Xiao, L., Zhou, L., Greiner, E., Fayer, R., 2004.
Prevalence and age-related variation of Cryptosporidium species and
genotypes in dairy calves. Vet. Parasitol. 122, 103117.
Santn, M., Trout, J.M., Fayer, R., 2008. A longitudinal study of cryptosporidiosis in dairy cattle from birth to 2 years of age. Vet. Parasitol. 155,
1523.
Santn, M., Trout, J.M., Fayer, R., 2009. A longitudinal study of Giardia duodenalis genotypes in dairy cows from birth to 2 years of age. Vet.
Parasitol. 162, 4045.
Starkey, S.R., Wade, S.E., Schaaf, S., Mohammed, H.O., 2005. Incidence of
Cryptosporidium parvum in the dairy cattle population in a New York
City watershed. Vet. Parasitol. 131, 197205.
Thompson, R.C.A., 2004. The zoonotic signicance and molecular epidemiology of Giardia and giardiasis. Vet. Parasitol. 126, 1535.
Thompson, H.P., Dooley, J.S., Kenny, J., McCoy, M., Lowery, C.J., Moore,
J.E., Xiao, L., 2007. Genotypes and subtypes of Cryptosporidium spp.
in neonatal calves in Northern Ireland. Parasitol. Res. 100, 619
624.
Trotz-Williams, L.A., Jarvie, B.D., Martin, S.W., Leslie, K.E., Peregrine, A.S.,
2005. Prevalence of Cryptosporidium parvum infection in southwestern Ontario and its association with diarrhea in neonatal dairy calves.
Can. Vet. J. 46, 349351.
Trout, J.M., Santn, M., Greiner, E., Fayer, R., 2004. Prevalence of Giardia
duodenalis genotypes in pre-weaned dairy calves. Vet. Parasitol. 124,
179186.
Trout, J.M., Santn, M., Greiner, E., Fayer, R., 2005. Prevalence and genotypes of Giardia duodenalis in post-weaned dairy calves. Vet. Parasitol.
130, 177183.
Trout, J.M., Santn, M., Greiner, E.C., Fayer, R., 2006. Prevalence and genotypes of Giardia duodenalis in 1-2 year old dairy cattle. Vet. Parasitol.
140, 217222.
Trout, J.M., Santn, M., Fayer, R., 2007. Prevalence of Giardia duodenalis
genotypes in adult dairy cows. Vet. Parasitol. 147, 205209.
Uehlinger, F.D., Barkema, H.W., Dixon, B.R., Coklin, T., OHandley, R.M.,
2006. Giardia duodenalis and Cryptosporidium spp. in a veterinary college bovine teaching herd. Vet. Parasitol. 142, 231237.
van Keulen, H., Macechko, P.T., Wade, S., Schaaf, S., Wallis, P.M., Erlandsen, S.L., 2002. Presence of human Giardia in domestic, farm and wild
animals, and environmental samples suggests a zoonotic potential for
giardiasis. Vet. Parasitol. 108, 97107.
Winkworth, C.L., Learmonth, J.J., Matthaei, C.D., Townsend, C.R., 2008.
Molecular characterization of Giardia isolates from calves and humans
in a region in which dairy farming has recently intensied. Appl. Environ. Microbiol. 74, 51005105.
Xiao, L., Fayer, R., 2008. Molecular characterisation of species and genotypes of Cryptosporidium and Giardia and assessment of zoonotic
transmission. Int. J. Parasitol. 38, 12391255.
Xiao, L., Feng, Y., 2008. Zoonotic cryptosporidiosis. FEMS Immunol. Med.
Microbiol. 52, 309323.
Xiao, L., Escalante, L., Yang, C., Sulaiman, I., Escalante, A.A., Montali, R.J.,
Fayer, R., Lal, A.A., 1999. Phylogenetic analysis of Cryptosporidium parasites based on the small-subunit rRNA gene locus. Appl. Environ.
Microbiol. 65, 15781583.