Infection, Genetics and Evolution 22 (2014) 229–234

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Infection, Genetics and Evolution

journal homepage: www.elsevier.com/locate/meegid

Genetic characterization of human-pathogenic Cyclospora cayetanensis

parasites from three endemic regions at the 18S ribosomal RNA locus
Irshad M. Sulaiman a,⇑, Ynes Ortega b, Steven Simpson a, Khalil Kerdahi a
a
Southeast Regional Laboratory, U.S. Food and Drug Administration, 60, Eighth Street NE, Atlanta, GA 30309, USA
b
Center for Food Safety, University of Georgia, 1109 Experiment Street, Griffin, GA 30223, USA

a r t i c l e i n f o a b s t r a c t

Article history: Cyclospora cayetanensis is an apicocomplexan parasite that infects the gastrointestinal tract and causes
Available online 24 July 2013 acute diarrheal disease in humans. In recent years, this human-pathogenic parasite has led to several
foodborne outbreaks in the United States and Canada, mostly associated with imported produce. Under-
Keywords: standing the biology and epidemiology of C. cayetanensis is difficult because little is known about its ori-
Gastro intestinal parasite gin, possible zoonotic reservoirs, and genetic relationships with other coccidian parasites. Recently, we
Foodborne disease developed a 70 kDa heat shock protein (HSP70) gene based nested PCR protocol for detection of C. caye-
Genetic marker
tanensis parasite and sequenced the PCR products of 16 human isolates from Nepal, Mexico, and Peru. In
Nucleotide sequencing
Rapid detection
this study, we have characterized the regions of 18S ribosomal RNA (rRNA) gene of 17 human C. cayetan-
ensis isolates for molecular detection, and also to ascertain the genetic diversity of this parasite. The 18S
rRNA primer sets were further tested by PCR amplification followed by nucleotide sequencing of the PCR
amplified products of previously characterized C. cayetanensis isolates from three endemic regions at
HSP70 locus. Although no genetic polymorphism was observed at the regions of HSP70 locus character-
ized in our previous study, the data analysis of this study revealed a minor genetic diversity at the 18S
rRNA locus among the C. cayetanensis isolates. The 18S rRNA gene-based nested PCR protocol provides
a useful genetic marker for the detection of C. cayetanensis parasite and confirms it as a genetically dis-
tinct species in genus Cyclospora. The results also supported lack of geographic segregation and existence
of genetically homogeneous population for the C. cayetanensis parasites both at the HSP70 as well as at
the18S rRNA loci.
Published by Elsevier B.V.

1. Introduction Cyclospora colobi and Cyclospora papionis were described from

Cyclospora cayetanensis is an emerging human-pathogenic api-
cocomplexan parasite that causes severe diarrheal disease in both
immunocompromised and immunocompetent individuals (Pape
et al., 1994; Turk et al., 2004). This organism was considered to
be a coccidian-like or cyanobacterium-like body in the beginning
(Ashford, 1979; Pollok et al., 1992; Bendall et al., 1993), and after-
wards it was confirmed to be a coccidian within the genus Cyclo-
spora based on oocyst morphological features (Ortega et al.,
1993). A number of mammalian and avian species have been
examined for the presence of C. cayetanensis, and humans have
been recognized to be the definitive host of this parasite. So far,
19 species of Cyclospora have been reported from various verte-
brate hosts based on conventional microscopic analysis (Lainson,
2005). Of these, three species such as Cyclospora cercopitheci,
⇑ Corresponding author. Address: Molecular Genetics Laboratory, Microbiological
Sciences Branch, U.S. Food and Drug Administration, 60, Eighth Street NE, Atlanta,
GA 30309, USA. Tel.: +1 404 575 1523.
E-mail address: irshad.sulaiman@fda.hhs.gov (I.M. Sulaiman).
primates. The morphologic and molecular characterizations
have revealed these three zoonotic species to be most related to
C. cayetanensis (Eberhard et al., 1999; Lopez et al., 1999).
In recent years, the C. cayetanensis parasite has been described
to cause several foodborne outbreaks in the United States and Can-
ada associated with imported produce (Ortega and Sanchez, 2010;
Hall et al., 2011). However, several other food types have also been
linked to cause the foodborne outbreaks. The transmission dynam-
ics of this parasite is still unknown. Consumption of contaminated
water sources as well as the fecal-oral route have been considered
as the primary means of spreading C. cayetanensis infection (Orlan-
di and Lampel, 2000; Ortega and Sanchez, 2010; Hall et al., 2011).
An epidemiologic study of C. cayetanensis infection in San Carlos Is-
land, Venezuela has revealed strong association between socio-
economic status and infection, and poverty has been considered
as an inclining factor for spreading the infection. Soil contaminated
with human feces has been suggested as the transmission route of
this parasite (Chacín-Bonilla et al., 2007; Chacín-Bonilla, 2008). The
biology and epidemiology of this human-pathogenic parasite is a
challenging task because attempts to grow in vitro or in animals

1567-1348/$ - see front matter Published by Elsevier B.V.

http://dx.doi.org/10.1016/j.meegid.2013.07.015
230 I.M. Sulaiman et al. / Infection, Genetics and Evolution 22 (2014) 229–234

have been unsuccessful. In addition, there is little known about
their origin, reservoirs, and genetic relationship with other related
parasites. Thus, oocysts in human feces are the only source for
morphological and molecular genetic characterizations of C. caye-
tanensis parasites (Ortega and Sanchez, 2010). In a previous study,
the phylogenetic analysis of 18S rRNA sequences indicated that the
human-associated Cyclospora species is closely related to members
of Eimeria genus (Relman et al., 1996).
Recently, we developed a two-step nested PCR protocol based
on the characterization of 70 kDa heat shock protein (HSP70) gene
for rapid detection of C. cayetanensis parasite (Sulaiman et al.,
2013). The validation results of these newly designed primers were
obtained by nucleotide sequencing of PCR amplified human C.
cayetanensis isolates from three different endemic regions that in-
clude Nepal, Mexico, and Peru (Sulaiman et al., 2013). This is a fol-
low up of our previous study. The present study characterizes the
18S ribosomal rRNA (rRNA) locus of C. cayetanensis compared with
the HSP70 locus to provide an additional genetic marker to en-
hance differentiation of isolates and improve accuracy of molecular
epidemiologic studies.
2. Materials and methods
2.1. Genomic DNA extraction
Fecal samples containing the oocysts of C. cayetanensis were ob-
tained from the infected individuals from three different geo-
graphic regions, and stored at 4 °C in 2.5% potassium dichromate
solution until utilized for DNA extraction (Table 1). Genomic
DNA was extracted from the purified oocysts using the QIAamp
DNA Stool Kit (QIAGEN Inc, Valencia, California) following the
manufacturer’s protocols. The concentration of purified genomic
DNA was measured at 260-nm absorbance using a NanoDrop-
1000 spectrophotometer (NanoDrop Technology, Rockland,
Delaware), and stored at –20 C until used to perform the PCR
and nucleotide sequencing.
2.2. Nested PCR
In order to amplify the 18S rRNA fragment of C. cayetanensis
parasite, a nested PCR protocol was developed using the primer
sets previously described by Li et al. (2007). For the primary PCR,
a fragment of 1000 bp was amplified using a forward (ExCycF,
50 -AAT GTA AAA CCC TTC CAG AGT AAC-30 ) and reverse (ExCycR,
50 -GCA ATA ATC TAT CCC CAT CAC G-30 ) primers. The 50 ll PCR
reaction mixture consisted of 25 ll of HotStart Taq Master Mix
(QIAGEN), and 25 ll of a solution containing 200 nM of each pri-
mer, 1.5 mM of additional MgCl2 (Promega, Madison, Wisconsin)
and template DNA (50 ng) diluted in PCR grade water. The QIAGEN
HotStart Taq Master Mix is a premixed solution containing Hot-
Start Taq DNA Polymerase, PCR Buffer, and dNTPs with a final con-
centration of 1.5 mM MgCl2 and 200 lM each dNTP. The reactions
were performed for 35 cycles (each cycle: 94 °C for 45 s, 55 °C for
45 s, and 72 °C for 60 s) in a GeneAmp PCR 9700 Thermocycler (Ap-
plied Biosystems, Foster City, CA), with an initial hot start (94 °C for
15 min) and a final extension (72 °C for 10 min). For the secondary
PCR, a fragment of 500 bp was amplified using 2.5 ll of primary
PCR reaction and a set of nested forward (NesCycF, 50 -AAT TCC AGC
TCC AAT AGT GTA T-30 ) and reverse (NesCycR, 50 -CAG GAG AAG
CCA AGG TAG GCR TTT-30 ) primers. The PCR conditions for the sec-
ondary PCR were identical to the primary PCR. The PCR products
were analyzed by agarose gel electrophoresis and visualized after
ethidium bromide staining. The genomic DNA of all the C. cayetan-
ensis isolates used in this study were amplified performing at least
two separate primary and secondary PCR reactions at the 18S rRNA
locus.
2.3. Nucleotide sequencing and data analysis
The secondary amplified 18S rRNA PCR products were se-
quenced using Applied Biosystems BigDye Terminator v3.1 Cycle
Sequencing Kit and an automated 3500 XL Genetic Analyzer
(DNA sequencer). The accuracy of generated DNA sequences was
confirmed by two-directional sequencing, and by performing addi-
tional sequencing of a new PCR product, if necessary. Multiple
alignments of nucleotide sequences were carried out using the Bio-
Edit, ClustalW, and Geneious programs with manual adjustment.
The generated nucleotide 18S rRNA sequences of various C. caye-
tanensis isolates were deposited in the GenBank database under
accession number KC662279–KC662295.
3. Results
Nucleotide sequences of the partial 18S rRNA gene (500 bp)
were generated for 17 human C. cayetanensis isolates belonging
to three geographically separated endemic regions located in Nepal,
Mexico, and Peru. Data analysis showed that the C. cayetanensis
18S rRNA sequences to be AT rich with minor variations ranging
from 54.52% to 54.72% for the region of this gene characterized
in the study. A similar trend of high AT-content was also noticed

Table 1
Cyclospora isolates used in the study, and their sequence type.
a
S. No. Isolate Country of origin Host Sequence type Reference (GenBank accession No.)
1. HMCCMX1 Mexico Human C. cayetanensis AF111183, This report
2. HMCCMX2 Mexico Human C. cayetanensis AF111183, This report
3. HMCCMX3 Mexico Human C. cayetanensis AF111183, This report
4. HMCCMX4 Mexico Human C. cayetanensis AF111183, This report
5. HMCCMX5 Mexico Human C. cayetanensis AF111183, This report
6. HMCCMX6 Mexico Human C. cayetanensis AF111183, This report
7. HMCCNP1 Nepal Human C. cayetanensis AF111183, This report
8. HMCCNP2 Nepal Human C. cayetanensis AF111183, This report
9. HMCCNP3 Nepal Human C. cayetanensis AF111183, This report
10. HMCCNP4 Nepal Human C. cayetanensis AF111183, This report
11. HMCCNP5 Nepal Human C. cayetanensis AF111183, This report
12. HMCCNP6 Nepal Human C. cayetanensis AF111183, This report
13. HMCCPR1 Peru Human C. cayetanensis AF111183, This report
14. HMCCPR2 Peru Human C. cayetanensis AF111183, This report
15. HMCCPR4 Peru Human C. cayetanensis AF111183, This report
16. HMCCPR5 Peru Human C. cayetanensis AF111183, This report
17. HMCCPR6 Peru Human C. cayetanensis AF111183, This report
a
Published C. cayetanensis 18S rRNA gene sequence; the above 17 human C. cayetanensis 18S rRNA gene sequences generated in this study
were submitted in the GenBank database under accession number KC662279–KC662295.
 I.M. Sulaiman et al. / Infection, Genetics and Evolution 22 (2014) 229–234
while analyzing the published full-length 18S rRNA sequence of C.
cayetanensis (52.70%, AF111183), and three other published rRNA
sequences of Cyclospora species obtained from primates that in-
clude C. cercopitheci (52.44%, AF111185), C. colobi (52.20%,
AF111186), and C. papionis (52.50%, AF111187).
To understand the genetic relationship between Cyclospora spp.
and member of apicomplexan species, the 18S rRNA sequences of
four Cyclospora species including C. cayetanensis (AF111183), C.
cercopitheci (AF111185), C. colobi (AF111186), and C. papionis
(AF111187) were aligned with published 18S rRNA sequences of
Babesia microti (JQ609304), Toxoplasma gondii (L37415), Cryptospo-
ridium baileyi (AF0934495), Cryptosporidium hominis (AF093491),
Cryptosporidium muris (AF093498), Cryptosporidium parvum
(AF093490), Cryptosporidium serpentis (AF093499), Eimeria acervu-
lina (FJ236372), Eimeria adenoeides (FR745913), Eimeria maxima
(FJ236351), Plasmodium falciparum (JQ627152), and Plasmodium vi-
vax (JQ627158) downloaded from GenBank, and a neighbor joining
tree was constructed (Fig. 1). The phylogenetic analysis clearly
showed formation of genus-specific clades. The four species of
Cyclospora formed a distinct clade in the tree with full statistical
reliability (bootstrap value, 100%). The rest of the apicomplexan
species examined also exhibited a similar trend with a significant
high bootstrap value (Fig. 1).
To measure the genetic polymorphism at the 18S rRNA locus,
between the C. cayetanensis isolates and among the members of
four Cyclospora species (C. cayetanensis, C. cercopitheci, C. colobi,
and C. papionis), and the evolutionary relationship of Cyclospora
parasites to other members of the phylum Apicomplexa (B. microti,
C. hominis, E. maxima, P. falciparum, and T. gondii), multiple se-
quence alignments were performed and evolutionary genetic dis-
tance was calculated for the generated 18S rRNA sequences
Fig. 1. Phylogenetic relationships of Cyclospora to other apicomplexan parasites at the 18S ribosomal RNA locus.
231
(Table 2). A distinct inter-species genetic variation was observed
throughout the 18S rRNA regions of the four species of Cyclospora
examined. The analysis further revealed a minor intra-species ge-
netic variation among the 17 generated 18S rRNA sequences of C.
cayetanensis. Of the 17 sequences, 13 of the sequences matched
100% with a published sequence of C. cayetanensis (AF111183), 3
of them (including HMCCMX2, HMCCMX2, and HMCCPR5)
matched 100% with a published sequence (U40261), and the se-
quence belonging to isolate HMCCPR2 matched about 99% with
the published AF111183 sequence (Table 2). Analysis of the data
revealed a closer genetic relationship among the four Cyclospora
species at the 18S rRNA locus. A significant close relatedness of
Cyclospora parasites were observed with E. maxima parasite,
whereas it was most distant with P. falciparum parasite (Table 2).
4. Discussion
Genetic characterization of human-pathogenic microorganisms
including gastrointestinal parasites has been widely successful in
developing rapid diagnostic tools, understanding their transmis-
sion dynamics, and in conducting epidemiologic investigations
associated with foodborne and waterborne outbreaks (Sulaiman
et al., 2003, 2004). The multi-locus sequence characterizations
have been effective for mapping the genetic polymorphism in spe-
cies with low genetic diversity, and in understanding the popula-
tion genetic structure of a species which lack geographic
segregation and possess some genetically isolated sub-populations
(Li et al., 2013). Furthermore, based on the nucleotide sequence
typing several unique apicomplexan species and genotypes known
to cause foodborne or waterborne outbreaks have been described
 232
Table 2
Evolutionary genetic distances among Cyclospora spp. and other organisms at the 18S rRNA locus.
a
Species Number of nucleotide differences/100 bases
C. cayetanensis C. cayetanensis C. cayetanensis C. cayetanensis C. colobi C. papionis C. cercopitheci E. maxima T. gondii C. hominis B. microti P. falciparum
HMCCMX2 HMCCMX4 HMCCPR2 AF11118 AF111186 AF111187 AF111185 FJ236351 L37415 AF093491 JQ609304 JQ627152

I.M. Sulaiman et al. / Infection, Genetics and Evolution 22 (2014) 229–234

C. cayetanensis 00.0
HMCCMX2
C. cayetanensis 99.8 00.0
HMCCMX4
C. cayetanensis 99.6 99.8 00.0
HMCCPR2
C. cayetanensis 99.8 100 99.8 0.0
AF111183
C. colobi 99.0 99.2 99.0 98.7 00.0
AF111186
C. papionis 98.6 98.8 98.6 98.6 99.4 00.0
AF111187
C. cercopitheci 98.4 98.6 98.4 98.4 99.4 99.2 00.0
AF111185
E. maxima 93.8 94.0 93.8 94.5 94.9 94.6 94.9 00.0
FJ236351
T. gondii L37415 84.7 84.9 85.1 87.5 87.9 87.6 87.5 86.2 00.0
C. hominis 71.7 71.5 71.7 80.3 80.3 80.4 80.4 79.3 82.6 00.0
AF093491
B. microti 75.7 75.7 75.7 81.5 81.7 81.7 81.4 80.4 81.6 80.2 00.0
JQ609304
P. falciparum 58.7 58.5 58.7 60.3 60.4 60.5 60.4 60.3 61.0 61.0 61.4 00.0
JQ627152
a
The values are nucleotide changes per 100 bp calculated by Kimura two-parameter method using the Geneious software.
 I.M. Sulaiman et al. / Infection, Genetics and Evolution 22 (2014) 229–234
(Morgan-Rayan et al., 2002; Sulaiman et al., 2002; Ryan et al., 2003,
2004).
Recently, we sequenced the region of HSP70 gene of C. cayetan-
ensis and identified a new biomarker for assessing the transmission
dynamics of this human-pathogenic parasite (Sulaiman et al.,
2013). Since HSP70 locus is conserved across the prokaryote and
eukaryote genomes, it has been widely used as a genetic marker
in developing rapid detection assays including in a number of api-
complexan species (Karlin and Brocchieri, 1998; Germot and Phi-
lippe, 1999; Schnittger et al., 2000; Sulaiman et al., 2000;
Yamasaki et al., 2002; Monteiro et al., 2008; Terkawi et al.,
2009). Sequence characterizations of HSP70 gene have also been
advantageous in understanding the genetic structure both at inter-
and intra- species level in closely related genera of Cyclospora such
as Cryptosporidium (Sulaiman et al., 2000). However, a complete
genetic homogeneity at the HSP70 locus was observed among
the 16 human C. cayetanensis isolates characterized from three
geographic locations (Sulaiman et al., 2013).
In the present study the 18S rRNA gene of 17 human C. cayetan-
ensis isolates from three different geographic locations was se-
quenced. Phylogenetic analyses of the 18S rRNA genes of various
published and generated isolates of C. cayetanensis (AF111183, this
study), C. cercopitheci (AF111185), C. colobi (AF111186), and C.
papionis (AF111187) indicated that the Cyclospora parasites have
multiple species; there is a significant interspecies diversity in
the genus Cyclospora. Results of phylogenetic analyses are largely
in agreement with our previous results obtained at HSP70 locus
(Sulaiman et al., 2013). However, the results of genetic analyses
indicated a minor intra-species polymorphism among the C. caye-
tanensis isolates from three geographic regions at the 18S rRNA lo-
cus (this study). Of the 17 human C. cayetanensis isolates
characterized, the 18S rRNA sequences of 13 isolates matched
100% with a published sequence (AF111183). However, the 18S
rRNA sequence belonging to isolate HMMMPR2 displayed a one-
point-mutation (C to T substitution) at position 110, and the rest
of the 3 sequences of HMCCMX2, HMCCMX3 and HMCCPR4
showed a one-point-mutation (C to T substitution) at position
119 as compared to the published sequence (AF111183). Using
the same 18S primer sets that was used in this study, another
group has also reported a similar result including presence of
low genetic diversity and two substitutions (C to T at position
110 and position 119) for the 35 C. cayetanensis isolates from China
characterized at the 18S rRNA locus (Zhou et al., 2011). The 18S
rRNA locus of C. cayetanensis has also been exploited for diagnos-
tics and epidemiologic surveys (Adam et al., 2000; Orlandi et al.,
2003; Ortega and Sanchez, 2010). Efforts are underway to charac-
terize a few more genetic loci to better understand the population
genetic structure and transmission dynamics of this human-path-
ogenic parasite.
5. Conclusions
In conclusion, the 18S rRNA gene is a good genetic marker for
the rapid detection of C. cayetanensis parasites. Sequence charac-
terization of C. cayetanensis parasites at the 18S rRNA locus, from
three different endemic regions (Nepal, Mexico, and Peru) showed
existence of low genetic diversity at the intra- species level. This
assay can be used to test food samples for the presence of C.
cayetanensis.
Acknowledgement
The findings and conclusions in this manuscript are those of the
authors and do not necessarily represent the views or official posi-
tion of the U.S. Food and Drug Administration (FDA). The names of
233
vendors or manufacturers are provided as examples of available
product sources; inclusion does not imply endorsement of the ven-
dors, manufacturers, or products by the FDA or the U.S. Depart-
ment of Health and Human Services. This study was supported in
part by funding from the Division of Field Sciences of FDA. The
funders had no role in the study design, data collection and analy-
sis, decision to publish, or preparation of the manuscript. Thanks
are also due to Nicky Sulaiman for her invaluable help in comple-
tion of this study and comments on this manuscript.
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