Veterinary Parasitology 196 (2013) 3136

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

A large scale molecular study of Giardia duodenalis in horses

from Colombia
Mnica Santn a, , Jess A. Corts Vecino b , Ronald Fayer a
a

Environmental Microbial and Food Safety Laboratory, ARS, US Department of Agriculture, Beltsville, MD, USA
Laboratorio de Parasitologa Veterinaria, Facultad de Medicina Veterinaria y de Zootecnia, Universidad Nacional de Colombia-Sede
Bogot, Bogot D.C., Colombia
b

a r t i c l e

i n f o

Article history:
Received 1 May 2012
Received in revised form 29 January 2013
Accepted 7 February 2013
Keywords:
Assemblage
Genotype
Giardia duodenalis
Horse
PCR
Prevalence

a b s t r a c t
The prevalence of Giardia duodenalis assemblages in horses is poorly known. The present
study examined feces from 195 horses, 1 month17 years of age, in 4 locations in Colombia.
Prevalence of infection was determined by PCR and all positives were sequenced to determine the genotypes. Thirty four (17.4%) horses were found positive. This is the rst report of
G. duodenalis in horses from Colombia. Prevalence in female and male horses was 18.9% and
15.1%, respectively. Prevalence in horses <1 year of age and horses >1 year of age was 21.1%
and 15.1%, respectively. Molecular characterization using the beta giardin (bg), glutamate
dehydrogenase (gdh), triose phosphate isomerase (tpi), and small subunit ribosomal RNA
(ssurRNA) genes identied G. duodenalis Assemblages A and B, the assemblages regarded as
zoonotic.
Published by Elsevier B.V.

1. Introduction
Giardia duodenalis (syn. G. lamblia, G. intestinalis) is
a widespread intestinal parasite of mammals. Molecular
characterization has identied seven major assemblages
(AG) of G. duodenalis that appear to have different host
ranges. Additional genotypes have been reported in mammals, genotype H in seals and the quenda genotype in
quenda and cattle (Adams et al., 2004; Lasek-Nesselquist
et al., 2010; Ng et al., 2011). Giardia was rst reported
as a parasite of horses in South Africa in 1921 (Fantham,
1921). Although it has been found in horses with diarrhea (Manahan, 1970; Kirkpatrick and Skand, 1985),
infected horses rarely show any clinical signs (Bemrick,
1968) and no subclinical effects have been reported.
Nearly all reports of giardiasis in equines are based on
microscopic studies in which cysts were observed in
feces (Fantham, 1921; Bemrick, 1968; Kirkpatrick and

Corresponding author. Tel.: +1 301 504 6774; fax: +1 301 504 6608.
E-mail address: monica.santin-duran@ars.usda.gov (M. Santn).
0304-4017/$ see front matter. Published by Elsevier B.V.
http://dx.doi.org/10.1016/j.vetpar.2013.02.006

Skand, 1985; Manahan, 1970; Xiao and Herd, 1994;

Pavlsek et al., 1995; Olson et al., 1997; Johnson et al.,
1997; Rigolon and Vargas, 1998; Atwill et al., 2000).
Recently, G. duodenalis Assemblages A, B, and E have
been detected in horses (Traub et al., 2005; Veronesi
et al., 2010). Because Assemblages A and B are known
to infect humans (Monis et al., 1999; Thompson, 2004)
horses could represent a reservoir of G. duodenalis with
the potential to cause disease in humans through direct
contact or by contamination of food and water supplies. Although molecular methods have recently been
applied to determine the prevalence of G. duodenalis
assemblages in some domesticated and companion animals (Feng and Xiao, 2011), information regarding the
prevalence of G. duodenalis in horses has been limited
to a very small number of animals (Traub et al., 2005;
Veronesi et al., 2010; Traversa et al., 2012). Therefore, the
present study was undertaken to determine the prevalence of G. duodenalis in a large number of horses, to
determine the assemblages present, and to determine any
age and gender related preferences in the prevalence of
infection.

DNA was extracted from each specimen using a DNeasy

Tissue Kit (Qiagen; Valencia, CA). The protocol utilized
proprietary reagents provided by the manufacturer with
modication. Fifty microliters of each cleaned fecal specimen was thoroughly mixed with 180 l of ATL buffer.
Twenty microliters of proteinase K (20 mg/ml) was added
and thoroughly mixed. After overnight incubation at 55 C,
200 l of AL buffer was added and the protocol then followed kit instructions with one exception. To increase the
DNA recovery, the nucleic acid was eluted in 100 l of AE
buffer.

33
41
24
24
122
1 (3.7)
5 (26.3)
0 (0)
5 (19)
11(15.1)
27
19
1
26
73
1 (3.1)
7 (14.6)
6 (25)
4 (27)
18(15.1)
32
48
24
15
119

No. positive (%)

No. positive (%)

1 (3.6)
7 (58.3)
0 (0)
8 (23)
16 (21.1)
28
12
1
35
76

No. positive (%)

No. positive (%)

Gender
Male
No. of horses
>1 year
No. of horses
Age
<1 year
No. of horses
No. positive (%)

2 (3.3)
14 (23.3)
6 (24)
12 (24)
34 (17.4)
60
60
25
50
195

2.3. DNA extraction

Total

To remove fecal debris and concentrate cysts, feces were

processed by sieving and CsCl density gradient centrifugation as described (Trout et al., 2004). Fifteen grams of feces
from each horse were thoroughly mixed with 35-ml dH2 O.
The suspension was passed through a 45 m pore size
wire mesh sieve, poured into a 50 ml screw top centrifuge
tube, and centrifuged at 1800g for 15 min. The supernatant
was aspirated, the pellet was thoroughly mixed in a 1:1
mixture of dH2 O:CsCl (1.4g/ml), and the suspension was
centrifuged at 300g for 20 min. The surface 4 ml of supernatant was aspirated, washed twice with dH2 O, and the
nal pellet was suspended in 500 l of dH2 O. The resulting
CsCl cleaned specimens were examined using molecular
methods as described below.

Location (region ID)

2.2. Concentration of cysts from feces

Table 1
Number of horses examined, number of horses positive, and prevalence (%) of Giardia duodenalis by age and sex at each location.

In 2008 fecal specimens were collected from 195 horses

in four regions in Colombia: 60 from Sabana de Bogot
(Region A), 60 from Costa Atlntica (Region B), 50 from
Llanos Orientales (Region C), and 25 from Bogot D.C.
(Region D) (Table 1). Region A horses are Silla Argentino
breed. Males are housed in separate stables and their drinking water is from the nearest municipally aqueduct while
females are pastured with colts with access to natural
spring water. Region B horses are Paso no colombiano.
Males are housed in separate stables while females are pastured with colts, and both males and females drink spring
natural water. Region C horses are almost wild with access
to spring natural water; they are only grouped together
once or twice a year for examination and care. Region
D horses at the Universidad Nacional de Colombia are
used for academic practices and have access to water from
the Bogota D.C. aqueduct. Overall, horses ranged from 2
months to 17 years of age and were represented by 73
males and 122 females. All horses appeared healthy. Feces
were collected directly from the rectum of each horse into
a plastic bag and each bag was marked to identify the horse.
Bags were sealed and immediately placed onto ice or cold
packs in an insulated container. Specimens were shipped to
the USDA laboratory in Beltsville, Maryland and processed
within 3 days after arrival.

Female
No. of horses

2.1. Animals and collection of specimens

Sabana de Bogot (A)

Costa Atlntica (B)
Bogot D.C. (C)
Llanos Orientales (D)
Total

2. Materials and methods

1 (3)
9 (22)
6 (25)
7 (29)
23(18.9)

M. Santn et al. / Veterinary Parasitology 196 (2013) 3136

No. of horses

32

M. Santn et al. / Veterinary Parasitology 196 (2013) 3136

2.4. PCR and DNA sequence analysis

33

obtained in this study have been deposited in GenBank

under accession numbers JX972180JX972187.

For each specimen a fragment of 292 bp of the small subunit ribosomal RNA (ssurRNA) gene was obtained by nested
PCR using primers a PCR protocol previously described
(Appelbee et al., 2003; Hopkins et al., 1997). DNA from
all ssurRNA positive specimens was subjected to PCR to
amplify fragments of the beta giardin (bg), glutamate
dehydrogenase (gdh), and triose phosphate isomerase (tpi)
genes. Nested PCRs using previously described primers
and PCR conditions were used to amplify fragments of the
tpi gene (Sulaiman et al., 2003) and the bg gene (Cacci
et al., 2002; Lalle et al., 2005). A semi-nested PCR was used
to amplify a fragment of the gdh gene using previously
described primers and PCR conditions (Read et al., 2004).
PCR products were analyzed on a 1% agarose gel and
visualized by staining the gel with ethidium bromide.
All positive PCR products were puried with Exonuclease
I/Shrimp Alkaline Phosphatase (Exo-SAP-ITTM ) (USB Corporation; Cleveland, OH) and sequenced in both directions
with the same internal PCR primers in 10 l reactions,
Big DyeTM chemistries, and an ABI 3130 sequencer analyzer (Applied Biosystems; Foster City, CA). For each gene,
sequence chromatograms of each strand were aligned and
examined with Lasergene software (DNASTAR; Inc., Madison, WI). The sequences were compared with sequences in
the GenBank database by BLAST. The nucleotide sequences

2.5. Statistical analysis

Fishers exact test was applied to data on the prevalence of infection in males versus females and to horses
less than one year of age versus those over one year of age.
Values in which p value was less than 0.05 were considered
signicant.
3. Results
3.1. Prevalence
Of fecal specimens obtained from 195 horses 34 (17.4%)
were PCR positive based on ssurRNA (Table 1). For horses
from regions A, B, C, and D, 3.3%, 23.3%, 24%, and 24% of
the specimens were found PCR positive for G. duodenalis,
respectively. These included 11 male and 23 female horses
that ranged in age from 2 months to 13 years (Table 2).
Prevalence of infection based on gender differences was
not signicant (p = 0.5624). Prevalence of infection based
on age differences was also not signicant (p = 0.3347).
Diarrhea was not observed in any of the specimens in the
present study.

Table 2
Assemblages of Giardia duodenalis determined by sequence analysis of ssurRNA, bg, gdh, and tpi genes for each positive horse are presented.
Horse ID

Location

Sex

Age

Assemblages
ssurRNA

17A
47A
4B
5B
10B
14B
15B
19B
20B
21B
22B
25B
30B
51B
53B
59B
1C
3C
9C
12C
24C
25C
12D
20D
33D
34D
35D
37D
38D
39D
42D
44D
46D
48D

Sabana de Bogot
Sabana de Bogot
Costa Atlntica
Costa Atlntica
Costa Atlntica
Costa Atlntica
Costa Atlntica
Costa Atlntica
Costa Atlntica
Costa Atlntica
Costa Atlntica
Costa Atlntica
Costa Atlntica
Costa Atlntica
Costa Atlntica
Costa Atlntica
Bogot D.C.
Bogot D.C.
Bogot D.C.
Bogot D.C.
Bogot D.C.
Bogot D.C.
Llanos Orientales
Llanos Orientales
Llanos Orientales
Llanos Orientales
Llanos Orientales
Llanos Orientales
Llanos Orientales
Llanos Orientales
Llanos Orientales
Llanos Orientales
Llanos Orientales
Llanos Orientales

Male
Female
Male
Male
Female
Female
Female
Female
Female
Female
Male
Female
Female
Male
Female
Male
Female
Female
Female
Female
Female
Female
Female
Female
Male
Male
Female
Female
Female
Female
Male
Male
Female
Male

7 months
18 months
5 months
8 months
4 years
10 years
12 months
15 months
7 months
7 months
16 months
13 years
4 years
3 months
2 months
8 months
6 years
10 years
9 years
9 years
4 years
2 years
9 months
8 years
11 months
3 years
8 years
13 months
10 months
7 months
3 months
2 months
3 months
5 months

Assemblage B
Assemblage B
Assemblage B
Assemblage A
Assemblage B
Assemblage B
Assemblage B
Assemblage B
Assemblage B
Assemblage B
Assemblage B
Assemblage B
Assemblage B
Assemblage B
Assemblage B
Assemblage B
Assemblage B
Assemblage B
Assemblage B
Assemblage B
Assemblage B
Assemblage B
Assemblage B
Assemblage B
Assemblage B
Assemblage B
Assemblage B
Assemblage B
Assemblage B
Assemblage B
Assemblage B
Assemblage A
Assemblage B
Assemblage B

bg

gdh

tpi

Assemblage A
Assemblage A

Assemblage B (B1)
Assemblage A

Assemblage B

Assemblage B (B1)
Assemblage B (B1)

Assemblage B (B1)
Assemblage B (B1)
Assemblage B (B1)

Assemblage B

Assemblage B (B1)

Assemblage B (B1)

Assemblage B (B1)

Assemblage B (B2)
Assemblage B (B1)
Assemblage B (B1)

Assemblage B (B1)
Assemblage B (B1)
Assemblage B (B2)

Assemblage B (B1)
Assemblage B (B1)

Assemblage B (B2)
Assemblage B (B2)
Assemblage B (B1)
Assemblage A
Assemblage B (B2)

Assemblage A (B1)
Assemblage B (B1)

Assemblage B

Assemblage B
Assemblage B
Assemblage B
Assemblage B

34

M. Santn et al. / Veterinary Parasitology 196 (2013) 3136

3.2. Sequence analysis

Sequence analysis of the 34 PCR positive samples
at the ssurRNA gene revealed the presence of G. duodenalis Assemblage A in 2 specimens (1.03% of 195
specimens) and Assemblage B in 32 samples (16.4% of
195 specimens). Nucleotide sequences identied as Assemblages A and B were identical to reference sequences
for those assemblages (Assemblage A: AY655700 and
Assemblage B: AF199447). Data from further testing
of DNA from these 34 PCR positive specimens using
primers for genes, bg, gdh, and tpi are shown in
Table 2.
Of 13 horses positive for the bg gene, 10 were identied as Assemblage B (two different nucleotide sequences
B1 and B2), and the other 3 were identied as Assemblage A (Table 2). All nucleotide sequences (Horses 4B,
5B, and 44D) identied as Assemblage A (subassemblage
AI) were identical to each other and had a 100% similarity (492/492 base pairs) with the reference sequence
(GenBank accession number XM 001705373). Of the 10
bg nucleotide sequences identied as Assemblage B, 9
were identical to one another (B1) (489 bp) and identical
also to nucleotide sequences JN416545 (dog from Croatia), JN416541 (dog from Croatia), JN416537 (dog from
Croatia), JF918480 (human from India), HQ616629 (lemur
from Spain), HQ179587 (human from Australia), FJ971466
(human from Thailand), FJ971438FJ971440 (humans from
Thailand) EU594668 (human from Cuba), and EU274393
(human from New Zealand). The other bg nucleotide
sequence (B2) (483 bp) isolated from Horse 12B was
compared by BLAST analysis to other sequences in GenBank database; it matched most closely with a 99.8%
similarity with nucleotide sequences HM165214 (human
from Sweden), AB618785 (human from Japan), FJ009209
(tamandua from Poland), EU637579 (human from Italy),
EU626199 (gazelle from Poland), EU637581 (macaque
from Italy), EU274389 (human from New Zealand) and
FJ009207 (human from Poland).
Fourteen horses were positive for the gdh gene, 12
were identied as Assemblage B (two different nucleotide
sequences B1 and B2) and 2 were identied as Assemblage A (Table 2). The nucleotide sequences were identied
for B1 in 8 horses and for B2 in 4 horses. When
nucleotide sequences B1 (423 bp) and B2 (423 bp) were
compared by BLAST analysis to other sequences in
the GenBank database, the closest match was 99.5%
(421/423 bp) and 99.3% (420/423 bp) similarity, respectively, with nucleotide sequences HM134212HM134214
(chinchilla from Brazil), HM134201 (brown howler
monkey from Brazil), L40508 (human from Australia),
EF507671EF507672 (human from Brazil), EF507646
(human from Brazil), AB618784, AY178738AY178739
(human from Australia), and AY826191(human from the
Netherlands). In two horses nucleotide sequences were
identied at gdh as Assemblage A. Those two sequences
(408 bp) were identical to AB159795 (ferret from Japan),
AB469364 (ferret from Brazil), AB508813 (ferret from
Japan), HM134217 (jaguar from Brazil), AB569380 (cat
from Japan), GQ426099 (bobcat from the USA), and
EF507642 (cattle from Brazil).

All nucleotide sequences obtained from 7 horses at the

tpi gene were identical to one another and were identied as Assemblage B (Table 2). When the tpi nucleotide
sequence (486 bp) was compared by BLAST analysis to
other sequences in the GenBank database, the closest
match was 99.8% (485/486 bp) with nucleotide sequences
GU564284 (human from China), EU637590 (mandrill from
Italy), AY368167 (wastewater from China), JQ363268
(wastewater from China), and AB569404 (human from
Japan).
One specimen (Horse 3) identied by ssurRNA, gdh, and
tpi as Assemblage B was identied by bg as Assemblage A.
4. Discussion
The results conrmed the presence of G. duodenalis in
horses from Colombia for the rst time. Diarrhea was not
observed in any of the specimens in the present study
and solid evidence for Giardia as a cause of illness in
horses is scant. Giardia infection was believed responsible for chronic diarrhea, weight loss, lethargy, inappetence,
and dermatitis in a 4-year-old Thoroughbred based on
the nding of cysts in feces and the resolution of clinical signs after treatment with metronidazole (Kirkpatrick
and Skand, 1985). Giardia was also one of many microorganisms listed as found in the diarrheic feces of race
horses in Sydney, Australia but no data were provided
regarding prevalence among affected horses, quantity of
cysts present, or effects of treatment on reduction or cessation of cyst excretion (Manahan, 1970). However, a survey
of 120 foals and 30 broodmares in Italy showed that Giardia infection did not correlate with the presence of diarrhea
in horses (Veronesi et al., 2010). Similarly, no association
between Giardia infection and the occurrence of diarrhea
was found in foals in a farm in South Ohio (Xiao and Herd,
1994).
In the present study thirty-four (17.4%) horses were
found G. duodenalis positive by PCR. Data obtained in
this study differ from most earlier studies of giardiasis in
equines which were based on microscopy to determine
prevalence or to report clinical signs and did not include
any molecularly based information (e.g. Fantham, 1921;
Bemrick, 1968; Kirkpatrick and Skand, 1985; Manahan,
1970; Xiao and Herd, 1994; Pavlsek et al., 1995; Olson
et al., 1997; Johnson et al., 1997; Rigolon and Vargas, 1998;
Atwill et al., 2000). The prevalence of G. duodenalis in fecal
specimens from horses has shown wide variation within
and between countries (035%) (Table 3). However, it is
difcult to compare prevalence data based on diverse diagnostic tests. Despite huge numbers of horses examined in
two surveys using coproscopical examination in Germany
(9192 and 4399 fecal samples) none were found Giardiapositive (Epe et al., 1993, 2004). However, Giardia cysts
were found in the same surveys in pigs, dogs, and cats. A
comparative study of the most commonly used microscopy
based detection tests (direct immunouorescence, fecal
otation using ZnSO4, and fecal smears stained with Lugol)
was conducted in Italy using fecal specimens from 120
foals and 30 broodmares (Veronesi et al., 2010). The prevalence obtained using fecal otation (2%) and stained fecal
smears (2%) was lower and in poor agreement with the

M. Santn et al. / Veterinary Parasitology 196 (2013) 3136

35

Table 3
Prevalence of Giardia duodenalis reported in horses.
Location
Brazil
Brazil
Brazil
Canada
Colombia
Czech Republic
Czech Republic
Germany
Germany
Germany
Italy

No. of animals
123
396
64
35
195
360
20
9192
37
4399
150

Age

Prevalence (% of positives)

Detection method

Reference

N/A
0>20 years
212 years
0>6 months
017 years

21.1
0.5
0
20
17.4

Rigolon and Vargas (1998)

de Souza et al. (2009)
Gomes et al. (2008)
Olson et al. (1997)
Present study

014 years
24 years
N/A
N/A
N/A
Foals: 0>8 weeks, mares:
N/A

5
35
0
5.4a
0
13.33 (DFA), 2 (fecal
otation and stained fecal
smears)

Microscopy
Microscopy
Microscopy
DFA
PCR and sequencing (tpi,
gdh, bg, and ssurRNA
Microscopy
Microscopy
Microscopy
Microscopy
Microscopy
DFA, fecal otation using
ZnSO4 , stained fecal smears
with Lugol, PCR and
sequencing (ssurRNA)
PCR and sequencing
(ssurRNA and tpi)
DFA

DFA and levitation

centrifugation test
DFA
DFA

Johnson et al. (1997)

Italy

431

N/A

8.6

USA

222

13

USA

91

Foals:124 weeks,
yearlings: >612 months,
mares: N/A
424 years

USA
USA

300
305

330 years
>2 years

0.66
4.6

Pavlsek et al. (1995)

Pavlsek et al. (1995)
Epe et al. (1993)
Beelitz et al. (1996)
Epe et al. (2004)
Veronesi et al. (2010)

Traversa et al. (2012)

Xiao and Herd (1994)

Forde et al. (1998)

Atwill et al. (2000)

N/A, information was not available; DFA, direct immunouorescence microscopy.

a
Prevalence reported in foals.

prevalence obtained by direct immunouorescence

(13.33%). In the present study, PCR was used to amplify
a fragment of ssurRNA to screen the samples. The same
PCR method used in the present study was used to
conrm those Giardia-positive samples observed by
direct immunouorescence microscopy for horses in Italy
(Veronesi et al., 2010). In the present study, and in most
published reports of Giardia in horses, the diagnosis was
conducted by examination of a single fecal sample per
horse. In a study that included multiple samples (311 fecal
samples) from 35 foals in Ohio the cumulative prevalence
for Giardia was 71%; with multiple positive samples found
in 52% of the Giardia infected foals (Xiao and Herd, 1994).
Given that cyst excretion in feces in that study was often
intermittent, the prevalence of Giardia reported in the
present study, which was based on a single sample per
horse, is probably underestimated.
The relationship between age of the horses and infection with G. duodenalis is not clear. In the present study,
prevalence of infection based on age differences was
not signicant. These ndings are similar to the ndings
reported for horses from KY and OH (Xiao and Herd, 1994)
and CA (Atwill et al., 2000). A Canadian study reported
a higher prevalence of infection in horses older than 6
months (25%) than in those less 6 months (0%) (Olson et al.,
1997). However, a survey in Italy found that prevalence of
Giardia was age related with infection being more common
in foals (23.33%) than adults (Veronesi et al., 2010).
To date, there are limited data on molecular characterization of isolates of G. duodenalis from horses. In
horses from ve Italian farms, all 20 positive isolates
of G. duodenalis were characterized at the ssurRNA gene
and identied as Assemblage E (Veronesi et al., 2010).
However, in another study in Italy that characterized 37
isolates using the ssurRNA and bg genes, 16, 11, and 6 were

identied as Assemblages A, B, and E, respectively (Traversa

et al., 2012). In another molecularly based study, isolates from 10 horses from New York State and Western
Australia were genetically characterized at the ssurRNA and
tpi genes; 6 isolates were identied as Assemblage B and
4 isolates were Assemblage A (Traub et al., 2005). In the
present study the presence of Assemblage B was more
prevalent than Assemblage A. In one horse (4B) isolate, it
was difcult to assign an assemblage because the genotyping result obtained at the bg locus was not consistent
with typing obtained at the other three loci (gdh, ssurRNA,
and tpi). The bg locus classied the isolate as Assemblage A
whereas the other three loci classied the isolate as Assemblage B (Table 3). Similar results have been reported in
studies also based on a multilocus approach showing a
number of human and animal isolates that could not be
unequivocally assigned at the assemblage level (Read et al.,
2004; Traub et al., 2004; Cacci et al., 2008). The inconsistent assemblage identication by the different genes could
be the result of recombination or the presence of a mixture with cysts of more than one assemblage in the fecal
specimen (Teodorovic et al., 2007; Beck et al., 2012).
The present study demonstrates that horses can be
infected with Assemblages A and B and therefore constitute
a potential zoonotic risk to humans. Because Giardia is well
known as a waterborne parasite contaminated water can
also serve as a source of infection. Wild horses and those
associated with agriculture and recreation can serve as a
source of human infection via watersheds

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