Veterinary Parasitology 108 (2002) 97107

Presence of human Giardia in domestic, farm and

wild animals, and environmental samples suggests a
zoonotic potential for giardiasis
Harry van Keulen a, , P. Timothy Macechko b , Susan Wade c ,
Stephanie Schaaf c , Peter M. Wallis d , Stanley L. Erlandsen b
a

Department of Biological, Geological and Environmental Sciences, Cleveland State University,

2399 Euclid Avenue, Cleveland, OH 44115, USA
b Department of Cell Biology and Neuroanatomy, School of Medicine,
University of Minnesota, Minneapolis, MN 55455, USA
Parasitology Section, Diagnostic Laboratory, Cornell University, P.O. Box 5786, Ithaca, NY 14852, USA
d Hyperion Research Ltd., 1008 Allowance Avenue, SE, Medicine Hat, AB, Canada T1A 3G8
Received 2 February 2002; received in revised form 2 July 2002; accepted 2 July 2002

Abstract
Giardia lamblia which parasitize humans belong to either of two genotypes, A or B, based on
specific signature sequences in the 5 end of the small subunit (16S) ribosomal RNA (rRNA) gene.
These two genotypes also were found in cysts from fecal samples of animal origin such as dogs,
cats, some farm animals and wild animals. In addition, trophozoites recovered from cysts obtained
from environmental samples belonged to these two genotypes as well, suggesting that the G. lamblia
genotypes A and B are widespread and possibly zoonotic. Trophozoites were recovered from rats
and these isolates might belong to another genotype of G. lamblia. Deer mice and one dog appeared
to be parasitized by genotypes of Giardia with close affinity to G. microti. This species, therefore,
also consists of a genotype complex.
2002 Elsevier Science B.V. All rights reserved.
GenBank accession numbers: Deer mouse Giardia, AF 473852; G. psittaci, AF 473853
Keywords: Giardia; Genotype characterization; Zoonosis

Corresponding author. Tel.: +1-216-687-4562; fax: +1-216-687-5443.

E-mail address: h.vankeulen@popmail.csuohio.edu (H. van Keulen).

0304-4017/02/$ see front matter 2002 Elsevier Science B.V. All rights reserved.
PII: S 0 3 0 4 - 4 0 1 7 ( 0 2 ) 0 0 1 8 1 - 4

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1. Introduction
The intestinal flagellate, Giardia, a common protozoan parasite of many mammals, is
the most commonly diagnosed cause of waterborne gastroenteritis in North America. Ingestion of cyst-contaminated surface water is probably the major cause of waterborne
outbreaks of Giardia. [See Thompson et al. (2000); Adam (2001) for reviews and major references.] Human giardiasis is caused by Giardia belonging to the morphological
group Giardia duodenalis as defined by median body shape (Filice, 1952). Within this
morphological group several species can be found: G. lamblia (syn. G. intestinalis), traditionally identified in humans, G. microti found in prairie voles and muskrats, G. ardeae
found in the blue heron and G. psittaci found in parakeets (Adam, 2001). G. lamblia exists as two major genotypes, which are referred to as Polish and Belgian, or assemblage A and B, respectively (van Keulen et al., 1995; Monis et al., 1999) and will here
be referred to as genotypes A and B. The question, whether giardiasis is a zoonosis
defined as a disease of animals transmissible to humans (Eckert, 1989)has been raised
in the past (Bemrick and Erlandsen, 1988). Although, person to person contact is a major
route of transmission, animal to person contact is also possible, implying that domestic,
farm and wild animals could act as carriers. Aquatic mammals (beavers and muskrats)
and rats are among the many possible animals that can contaminate drinking water reservoirs and have the potential to infect large numbers of people (Bemrick and Erlandsen,
1988). However, muskrats and prairie voles can be excluded because they harbor a unique
Giardia species, G. microti, as shown by sequence analysis of the 16S-ribosomal DNA
(rDNA) (van Keulen et al., 1998). However, other animals still could function as a source of
infection.
This study addresses whether dogs, cats and certain farm animals (cows, pigs and sheep)
are possible carriers of G. lamblia. The method used was rDNA typing by a combination
of polymerase chain reaction (PCR) and PCR followed by restriction enzyme digestion
(restriction fragment length polymorphism, RFLP) and DNA sequence analysis. The results
show that G. lamblia of genotypes A and B, can parasitize domestic and farm animals. In
addition, we found that deer mice and a dog isolate were parasitized by different Giardia
genotypes.

2. Materials and methods

2.1. Sample origin and preparation
Samples were obtained from several laboratories and had different origins and treatments.
Samples from various places throughout Canada were obtained from sewage and water
sediments, and retrieved into axenic culture after passage through gerbils (Wallis et al.,
1996). Pellets of these cultured Giardia trophozoites were shipped frozen and stored at
70 C until use. A total of 14 different samples was analyzed (Wallis et al., 1996). In
addition, 12 human samples, 1 sample of filtered water and 1 beaver sample (formalin fixed
crude samples) obtained from a waterborne outbreak in Ontario (the town of Temagami)
were analyzed. Other samples of animal origin were crude fecal samples or sucrose purified

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99

cysts. Most domestic and wild animal samples came from the State of New York but some
dog and cat isolates came from Minnesota. These were evaluated by light microscopy
and subsequently removed from the slides and suspended in approximately 1 ml of water.
Trophozoites from intestinal scrapings from rats (Rattus norvegicus) obtained at the St.
Croix river in Minnesota, deer mice (Peromyscus maniculatus) and parakeets were isolated
by differential binding to plastic dishes in phosphate buffered saline as described (Feely
and Erlandsen, 1981; van Keulen et al., 1998).
2.2. DNA techniques
Isolation of DNA from cultured trophozoites was by standard procedures including proteinase K digestion and phenol extractions as described (Campbell et al.,
1990). The isolation of DNA from crude fecal samples containing cysts and environmental samples for PCR was by boiling the samples in 2.5 M NaCl at 95 C for 15 min,
followed by phenolchloroformisoamylalcohol extractions (50:48:2 (v/v)) and ethanol
precipitation. PCR was performed under conditions described previously (Weiss et al.,
1992). The primers used were forward primer 1: 5 -TCCGGTGGATCcTGCCGGA-3
(321) which contains a one base mismatch (lower case) to provide a BamHI site
(underlined) and reverse primer A: 5 -GCTCTCCGGAGTCGAAC-3 (285301) which
provides a BsiMI site (underlined) for cloning or primer K: 5 -TGGCGGCGGGGtaCCTTC3 (510528) which contains a two base mismatch and provides a KpnI site for cloning.
Genotype specific primers were modifications of primer 1: genotype A specific; 5 GGTGGATCCTGCCGGAGCG-3 (624) and genotype B specific; 5 -GGTGGAT
CCTGCCGGAATC-3 . The underlined bases indicate the genotype specific signature sequences. The entire 16S-ribosomal RNA (rRNA) gene from deer mouse Giardia
DNA was amplified with primers 1 and reverse primer R39: 5 -CCCGGGATCCAAGCTTGATCCTTCTGCAGGTTCACCTAC-3 , position 14251448. Amplification was
assessed by electrophoresis of 15100% of the PCR samples on 6% polyacrylamide gels
or 1% agarose gels, depending on the expected sizes of the amplified DNAs. Subsequent cloning work was in plasmid pUC19 restricted with BamHI + AvaI for the 300 bp
fragments and BamHI + KpnI for the 500 bp fragments (van Keulen et al., 1995; van
Keulen et al., 1998). For PCRRFLP analysis the amplified DNA was purified by one
phenolchloroform extraction and two ethanol precipitations. The DNA was subsequently
digested with HhaI.
2.3. DNA sequence analysis
Double stranded DNA sequencing reactions were performed in both directions using
Sequenase (Amersham Pharmacia Biotech Inc., Piscataway, NJ) and the protocols provided
by the manufacturer. Because band compressions occurred in these sequences, most were
analyzed on 8% polyacrylamide/8 M urea/20% formamide gels. Sequence alignments of
16S-rDNA was performed by using ClustalW (DNAStar, Madison, WI) in the Lasergene
package and ClustalX (http://www-igbmc.u-strasbg.fr/BioInfo). The latter programs also
were used to construct a phylogenetic tree and to perform bootstrap analysis (Thompson
et al., 1994).

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3. Results
3.1. Amplification of Giardia DNA from environmental samples
Giardia species in environmental samples obtained from various locations in Canada
were identified by PCR to establish whether the organisms belonged to genotype A or B.
The DNA was purified from trophozoites or fecal samples and the specific sense primers
that discriminate between genotypes were used. In this manner of the 14 Canadian samples, 10 were identified as genotype A while 4 were of the B type. Controls of known
genotypes A and B showed high specificity of the primers. Although, these type-specific
primers could discriminate between each genotype, they could not clearly identify different
species because they would score G. ardeae as genotype A and G. microti as genotype B.
In addition, they would not identify G. muris, which DNA does not amplify with these
primers. Although, these species have not been observed in any Giardia samples of human
origin (van Keulen et al., 1995), a clear identification of all species and genotypes will be
important to type samples from the environment or animals. Inspection and comparison of
the rDNA sequence between position 1 and 299 of all different Giardia species revealed that
digestion with the restriction enzyme HhaI would give a characteristic pattern of this DNA
segment enabling the use of restriction fragment length polymorphism (RFLP) analysis. An
example of the restriction patterns for five different Giardia species and several genotypes
are shown in Fig. 1. PCR amplified DNA from different Giardia species using the universal
primers 1 and A (verified by DNA sequence analysis) was purified, digested with HhaI and
characterized by gel electrophoresis. All five genotypes: G. lamblia genotype B and A, G.
microti, G. muris and G. ardeae, from left to right in Fig. 1, gave a distinct RFLP pattern. To
determine whether G. lamblia DNA or that from other Giardia species was present in environmental samples from Canada, the amplified DNA, using universal Giardia primers 1 and
A were purified and digested with the restriction enzyme HhaI. The PCRRFLP analysis
showed that all samples earlier identified as genotype A or B were correctly identified as
such and that no other species of Giardia than that of G. lamblia were among these samples.
Another 10 samples from patients from one outbreak of giardiasis in Ontario, Canada, were
analyzed in the same manner. Of the 10 samples that were tested, 4 could not be amplified;
the remainder were all of genotype A. A total of 32 samples from cultured Giardia isolates
were tested, of these 21 belonged to genotype A and 11 to genotype B.
3.2. Analysis of dog and cat Giardia
A number of isolates from animals were tested as described earlier. Only genotypes A and
B of G. lamblia were encountered among dog and cat samples. The results are summarized
in Table 1. A representative analysis of animal isolates is shown in Fig. 2. To investigate
whether any sequence variants were present, all samples were amplified with the primers 1
and K for subsequent cloning and sequence analysis. In all cases, the results of PCRRFLP
were confirmed by sequence analysis and no other sequences were encountered other than
those of the typical G. lamblia genotypes A and B. A total of eight dog and six cat samples
belonged to genotype A, while another eight dog and three cat samples belonged to genotype
B. A number of Giardia samples from farm animals (cow, sheep and pig) were tested by

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Fig. 1. PCRRFLP analysis of Giardia genotypes. DNA was amplified from plasmid clones of all species and
genotypes indicated, using primers 1 and A. The cloned sequences were first verified by DNA sequence analysis.
Amplified DNA was purified and digested with HhaI and size fractionated on an 8% polyacrylamide gel. The
DNA size marker was HaeIII digested pUC19, lane 1 (sizes in basepairs). The amplified and digested DNAs were
obtained of clones with the 5 298 bp of 16S-rDNA from G. lamblia genotype B, lane 2; G. lamblia genotype A,
lane 3; G. microti (prairie vole isolate), lane 4; G. muris, lane 5; and G. ardeae, lane 6.

PCRRFLP and, except for one calf sample (genotype B), were shown to belong to the
G. lamblia genotype A. This was also true for samples from two different groundhogs, a
bobcat and a prairie dog.
3.3. A different rDNA sequence in a Giardia sample from a dog
Samples from a litter of puppies (a greyhound/labrador cross, isolate 932036) from the
Department of Veterinary Medicine at Cornell University showed a different PCRRFLP
pattern. The amplified DNA was isolated, cloned and subjected to sequence analysis. A
number of base changes not present in the standard G. lamblia 16S-rDNA sequence were
observed. Some of the bases were identical to those seen in prairie vole and muskrat isolates;
however, the typical G. microti signature sequence (van Keulen et al., 1998) was not present.
The 5 298 nucleotides of this sequence are shown in Fig. 3 aligned with those of all other Giardia genotypes described here. The sequence of this dog isolate is identical to that of an Australian dog sample from group 4, isolates D6 and D46, described by Hopkins et al. (1997).

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Fig. 2. PCRRFLP of DNA from animal isolates. DNA was purified from trophozoites (controls) and cysts (animal
isolates) and amplified with primers 1 and A. The amplified DNA was purified and digested with HhaI. The digested
DNA was size fractionated on an 8% polyacrylamide gel. The 1 kbp ladder DNA (Gibco/BRL, Grand Island, NY)
was used, the sizes of the small fragments are indicated in basepairs. DNA was amplified from G. ardeae, lane 1;
G. muris cysts, lane 2; G. lamblia genotype B, dog (named woof) isolate, lane 3; G. microti, isolate prairie vole
11, lane 4; G. lamblia genotype A, isolate Be-J, lane 5; cat 933683, lane 6; prairie dog, lane 7; bobcat, lane 8;
groundhog, lane 9; hamster, lane 10; cat 926407, lane 11; calf, lane 12.

3.4. The rDNA sequence from a deer mouse isolate

Amplification and analysis of DNA isolated from several deer mice (P. maniculatus)
showed a different PCRRFLP pattern than that of other Giardia species. Therefore, the
entire 16S-rRNA gene from one sample from a deer mouse was amplified, cloned and
sequenced. The 16S-rDNA sequence was different from all other Giardia rDNA sequences,
and although, it contained the typical signature sequence of the genotype B (ATC, position
22) and the typical G. microti signature sequence GCCCCC, position 411), many other
positions were different. This suggests that deer mice are parasitized by yet another Giardia
species or genotype, distinct from that found in prairie voles and muskrats. The sequence
is 3.2% different from that of G. microti and 4.5% from that of genotype A of G. lamblia.
The 5 298 bases are shown in Fig. 3. The entire sequence was submitted to GenBank and
has accession number AF473852.
Since rats and humans inhabit the same habitat and rats often are found in sewers, a number
of rat samples (Rattus norvegicus) were obtained and analyzed by DNA amplification and
sequence analysis. The sequence of the 5 298 bases is presented in the alignment in Fig. 3. In
three different rat samples, a number of positions were consistently different when compared
to G. lamblia genotypes A and B. For instance, an A in position 62 (numbering based on

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Fig. 3. Sequence alignment 5 end 16S-rDNA. The alignment of the 5 298 bases of all species and genotypes
belonging to the morphological group G. duodenalis and G. muris are shown. The sequences were aligned using
ClustalX. The sequence of the dog isolate is from dog 932036 and is given on the top line. Identical bases are
indicated with a dot (), deletions to optimize alignment with a dash ().

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Table 1
Overview of characterized Giardia lamblia isolates from stool samples
Genotype A
Human 114a
Pigb
Cow 1c
Cow umpire6c
Calf 45c
Sheep 940901c
Prairie dog 946791c
Bobcat 960816.1c
Groundhog wc4309c
Groundhog 951608c
WW-32d

Genotype B
Cat 797475c
Cat Winniec
Cat 933683c
Cat 926407c
Cat 9501c
Cat 954957c
Dog 1e
Dog 2e
Dog 936564c
Dog 843803-2c
Dog 965650c
Dog 953942c
Dog 925872c
Dog 990887c

Dog D95c
Dog A796955c
Dog woofe
Dog 615c
Dog D911734c
Dog 901967c
Dog 991642c
Cat 799195c
Cat 954977c
Cat 975957c
Calfe

The isolates are marked with their name or isolation number. The data are based on sequence information obtained
by PCR of DNA directly purified from fecal material.
a The origin was Cleveland MetroHealth Medical Center.
b The origin was Czech Republic.
c Collected at veterinary school, Ithaca, NY, from various locations in New York State; the prairie dog sample
came from the San Francisco Zoo.
d From sewage samples (New York city watershed).
e Collected at The University of Minnesota from in and around Minneapolis.

Fig. 4. Phylogenetic tree of G. duodenalis species and genotypes. A phylogenetic tree was constructed using the
alignment shown in Fig. 3. The sequence of G. muris was used as an outgroup. The branching was supported by
bootstrap analysis (1000 replicates) and the values are indicated. The number of base changes per 100 is indicated
by the bar.

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105

G. lamblia genotype A) is found in many Giardia species, but this position is always a T in
genotype A and a G in genotype B.
3.5. Sequences of all G. duodenalis genotypes compared
The sequence of the 16S-rRNA gene of Giardia isolated from deer mice is unlike that of G.
muris and it belongs clearly to the morphologically-based G. duodenalis group of sequences.
This agrees with morphological observations (Erlandsen, unpublished observations). This is
also true for Giardia psittaci found in parakeets, which belongs to the G. duodenalis group,
based on the median body structure (Erlandsen and Bemrick, 1987). Genomic DNA was
isolated from G. psittaci and amplified with primers 1 and K. The DNA sequence obtained
was 500 bp of which the 5 298 is presented in Fig. 3 and compared to that of other isolates.
Clearly, the sequence is distinct from that of other Giardia species. All 5 298 bp sequences
of the various isolates described earlier belonging to the G. duodenalis group were aligned
and a phylogenetic tree was constructed, using the sequence of G. muris 16S-rDNA as
outgroup. The tree obtained using the neighbor-joining method and supported by bootstrap
analysis is shown in Fig. 4.

4. Discussion
It is clear from these results that the isolates belonging to genotype A and B of G. lamblia
are widespread in nature and can be found in humans, farm animals, pets and some wild
animals, and appear in the environment as well. This strongly raises questions concerning
the origin of giardiasis, in particular infection with genotypes A and B, which could be
zoonotic. However, it is not clear whether animals act as a reservoir host for humans or vice
versa, as was also discussed by Thompson et al. (2000). Giardia from parakeets, deer mice,
prairie voles and muskrats are clearly different, but still belong to the morphological group
duodenalis as defined by Filice (1952). This group is not confined to G. lamblia only, as
suggested by Thompson et al. (2000). That Giardia from parakeets belongs to a different
species, G. psittaci, is now supported by sequence analysis of part of the 16S-rDNA. As
shown before (van Keulen et al., 1998), the G. microti group is quite heterogeneous, and
consists of several genotypes, which form a cluster of related sequences, distinct from that of
G. lamblia. It appears that the deer mouse isolate belongs to the G. microti genotype cluster.
This was also the case for an isolate from one dog. It appears that both G. microti and G.
lamblia each exist as a complex of related groups, assemblages or genotypes. Although,
the tree shown in Fig. 4 was based on the alignment of 298 bases, to be able to include
the sequence of the rat isolate, the tree topology remained the same when 500 bases were
used for the other isolates. The rat isolate appears to belong clearly to the G. lamblia
genotype cluster. Whether rats act as reservoirs is not clear since no human samples have
been identified with these particular base changes.
The presence of a microti-like sequence in a dog sample and a microti-like sequence
in deer mice suggests that this species might not be specific for only prairie voles and
muskrats. As shown previously (van Keulen et al., 1998), this group shows a high degree of
variation. The one vole and two muskrat samples used in the alignment were the most distant

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sequences found. The microti group is clearly a heterogeneous group and host specificity
might not be strong as also appears to be the case for the lamblia group. With respect
to the dog isolate, it is striking that exactly the same sequence was found in two dogs in
Australia, named D6 and D46, which form a group 4 in the work of Hopkins et al. (1997).
The study by the same group showed that isolates from humans and dogs, living in a close
community, were different. The human isolates in this study belonged to groups identical
to genotypes A and B, but the dog samples were different. A similar observation was made
by Monis et al. (1998) who showed that dog isolates belonged to different assemblages of
G. lamblia, but control samples taken from Canadian and European dogs belonged to the
typical A and B genotypes of G. lamblia, as was shown here. Finally, the samples obtained
from three cows appeared to belong to the genotype A (2) or B (1) of G. lamblia and not to
the more abundant hoofed livestock genotype as described by Monis et al. (1999), which
were shown to be more prevalent in samples from calves from both Western Canada and
Australia (OHandley et al., 2000). However, the present sample was too small to derive
any significant conclusions, but suggests that cows and other farm animals produce cysts
potentially infective for humans.
In conclusion, the G. lamblia genotypes A and B were found in human, animal and
environmental samples. Isolates from rats might belong to another genotype of G. lamblia.
Prairie voles, muskrats and deer mice appeared to be parasitized by different genotypes
within the G. microti complex.

Acknowledgements
This work was supported by the Ohio Board of Regents through the Established Full-Time
Faculty Research Development Program.
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