1

REVIEW ARTICLE

Moving towards an integrated approach to molecular

detection and identification of Toxoplasma gondii

C. SU 1*, E. K. SHWAB 1, P. ZHOU 2, X. Q. ZHU 2 and J. P. DUBEY 3

1
Department of Microbiology, The University of Tennessee, Knoxville, TN 37996, USA
2
Laboratory of Parasitology, College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642,
China
3
Animal Parasitic Diseases Laboratory, United States Department of Agriculture, Beltsville, MD 20705, USA

(Received 5 May 2009; revised 9 June and 5 July 2009; accepted 6 July 2009; first published online 21 September 2009)

SUMMARY

The development of simple, sensitive and rapid methods for the detection and identification of Toxoplasma gondii is
important for the diagnosis and epidemiological studies of the zoonotic disease toxoplasmosis. In the past 2 decades,
molecular methods based on a variety of genetic markers have been developed, each with its advantages and limitations.
The application of these methods has generated invaluable information to enhance our understanding of the epidemi-
ology, population genetics and phylogeny of T. gondii. However, since most studies focused solely on the detection but not
genetic characterization of T. gondii, the information obtained was limited. In this review, we discuss some widely used
molecular methods and propose an integrated approach for the detection and identification of T. gondii, in order to
generate maximum information for epidemiological, population and phylogenetic studies of this key pathogen.

Key words: Toxoplasma gondii, genetic characterization, molecular detection and identification.

INTRODUCTION spread and cause severe damage to the foetus. In

immunocompromised patients, the reactivation of a
Toxoplasma gondii is an obligate intracellular, api-
latent infection can cause life-threatening encepha-
complexan parasite that infects all warm-blooded
litis (Montoya and Liesenfeld, 2004).
vertebrates, including mammals and birds. It is the
T. gondii has subpopulation structures in different
only known species in the genus Toxoplasma and is
geographical regions. It has what appears to be a
considered to be one of the most successful eukaryotic
clonal population structure in North America and
pathogens in terms of the number of host species
Europe, with 3 predominant lineages (named types
and percentage of animals infected worldwide. Up to
I, II and III) defined by multi-locus enzyme
one-third of the human population in the world
electrophoresis (MLEE), polymerase chain reaction-
is chronically infected (Dubey and Beattie, 1988 ;
restriction fragment length polymorphism (PCR-
Tenter et al. 2000). Human infections are primarily
RFLP) or microsatellite analysis (Darde et al. 1992 ;
caused by ingesting undercooked meat containing
Howe and Sibley, 1995 ; Ajzenberg et al. 2002 a). A
viable tissue cysts or by ingesting food or water
recent report suggests the same clonal structure in
contaminated with oocysts shed in the faeces from
Africa (Velmurugan et al. 2008). However, T. gondii
infected cats (Dubey, 2004). Primary infections in
isolates from animals and human patients in South
adults are mostly asymptomatic but lymphadeno-
America are diverse and largely distinct from those
pathy or ocular toxoplasmosis can occur in some
in North America and Europe (Ajzenberg et al.
patients. Severe acute, disseminated toxoplasmosis
2004 ; Khan et al. 2006 ; Lehmann et al. 2006 ; Pena
can occur in immunocompetent human patients
et al. 2008 ; Dubey et al. 2008 d). Historically, these
when infected with some isolates (Bossi and Bricaire,
distinct and non-type I, II or III parasites were
2004). Infection acquired during pregnancy may
designated as the ‘ atypical ’ or ‘ exotic ’ isolates. The
low linkage disequilibrium among genetic loci of
these isolates suggests that the parasites have under-
gone frequent sexual recombination (Ajzenberg et al.
* Corresponding author : Department of Microbiology,
the University of Tennessee, Knoxville, TN 37966, USA. 2004 ; Lehmann et al. 2004 ; Su et al. 2006).
Tel: +865 974 4015. Fax: +865 974 4007. E-mail : The consequences of infection with T. gondii
csu1@utk.edu depend on the host species and parasite genotypes.

Parasitology (2010), 137, 1–11. f Cambridge University Press 2009

doi:10.1017/S0031182009991065 Printed in the United Kingdom

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 C. Su and others 2

In mice, type I lineages are uniformly lethal Molecular detection by conventional PCR, nested
(LD100=1) ; by contrast, the type II and III lineages PCR (n-PCR) or quantitative real-time PCR
are significantly less virulent (LD100>=103) (Sibley (qPCR) of repetitive DNA sequences
and Boothroyd, 1992). In humans, disease manifes-
tations vary widely, ranging from asymptomatic to To achieve high sensitivity, PCR of a small, repeti-
severe acute toxoplasmosis (Bossi and Bricaire, tive DNA sequence is preferred, because the ef-
2004 ; Montoya and Liesenfeld, 2004). Type II is the ficiency of amplifying a small DNA fragment is
predominant lineage causing human toxoplasmosis. higher than that of a large one. In addition, there
However, there are biases between disease pres- are more template copies in a repetitive sequence
entations and parasite genotypes. For examples, type per organism. Three repetitive DNA sequences are
I or type I-like atypical isolates are more likely to be often used for the detection of T. gondii in biological
involved in severe toxoplasmic retinochoroiditis in samples, including the 35-copy B1 gene, the 300-
human patients (Grigg et al. 2001) and the atypical copy 529 bp repeat element and the 110-copy in-
isolates often cause severe acute, disseminated tox- ternal transcribed spacer (ITS-1) or 18S rDNA gene
oplasmosis in immunocompetent patients (Bossi and sequences. A molecular detection method (conven-
Bricaire, 2004). tional PCR) for T. gondii, targeting the B1 gene, was
Clinical symptoms of T. gondii infection are first developed by Burg et al. (1989). This method
non-specific and unreliable for diagnosis. The con- has since been modified and adapted in different
ventional diagnosis of T. gondii infection usually laboratories (Khalifa et al. 1994 ; Liesenfeld et al.
employs serological tests, bioassays in cats and/or 1994 ; Bretagne et al. 1995 ; Pelloux et al. 1998 ;
mice, or a combination of the 2 approaches (Dubey Contini et al. 2002, 2005 ; Reischl et al. 2003 ; Switaj
and Beattie, 1988). In the past 2 decades, the diag- et al. 2005 ; Bastien et al. 2007). The 529 bp repeat
nosis of T. gondii infection by direct detection of element was identified by Homan et al. (2000), and it
parasite-specific DNA in biological samples using was reported to be 10- to 100-times more sensitive
PCR-based molecular methods has gained popu- than the B1 gene (Homan et al. 2000 ; Reischl et al.
larity. The molecular diagnosis is more sensitive 2003 ; Calderaro et al. 2006). The 110-copy ITS-1 or
and cost-effective than the conventional methods 18S rDNA has been used as the target in a few
(Schoondermark-Van De Ven et al. 1994 ; Bessières studies (Hurtado et al. 2001 ; Jauregui et al. 2001 ;
et al. 2009). There are hundreds of papers in current Calderaro et al. 2006) and showed similar sensitivity
literature databases (such as PubMed) describing to the B1 gene. To achieve higher sensitivity, n-PCR
the use of these molecular methods. However, in this of B1 gene and ITS-1 sequences has been applied in
review article, rather than conduct an exhaustive some studies (Pelloux et al. 1998 ; Hurtado et al.
review of all of the literature, we focus on key, rep- 2001 ; Jauregui et al. 2001 ; Contini et al. 2002, 2005 ;
resentative articles to provide a perspective on es- Bastien et al. 2007), both markers showed high
tablishing an integrated approach for the detection sensitivity, with the detection level being as low as 1
and characterization of T. gondii. parasite (Hurtado et al. 2001 ; Jauregui et al. 2001 ;
Calderaro et al. 2006). For a given repetitive se-
quence, n-PCR is more sensitive than conventional
MOLECULAR METHODS FOR THE DETECTION
PCR. A case in point is that the n-PCR of the B1
AND IDENTIFICATION
gene is more sensitive than the conventional PCR for
Molecular methods rely on PCR for the specific de- the detection of T. gondii in amniotic fluid samples
tection or analysis of T. gondii DNA. These methods from congenital toxoplasmosis of human (Okay et al.
have proved to be simple, sensitive, reproducible 2009). Recently, we employed a set of primers for
and cost-effective, and have been applied to a variety n-PCR of T. gondii 18S rDNA, and found that it was
of clinical samples from animals and humans (Bell more sensitive than the B1 gene (data not shown).
and Ranford-Cartwright, 2002 ; Contini et al. 2005 ; The 18S rDNA marker is of particular interest, as it
Calderaro et al. 2006 ; Bastien et al. 2007). Molecular can distinguish several protozoan parasites which are
methods can be divided into 2 groups. The first closely related to T. gondii. There is further dis-
group focuses on specific detection of T. gondii DNA cussion of this marker in a later section (T O W A R D A N
in biological samples. The conventional PCR, nested INTEGRATED APPROACH FOR MOLECULAR DE-
PCR (n-PCR) and quantitative real-time PCR TECTION AND IDENTIFICATION) of this article.
(qPCR) of repetitive DNA sequences belong to this In recent years, qPCR that targets the repetitive
group. The second group of molecular methods DNA sequences, has shown high sensitivity (de-
focuses on a high resolution identification of T. gondii tecting y1 parasite genome equivalent) (Jauregui
isolates. The multilocus PCR-RFLP, microsatellite, et al. 2001 ; Reischl et al. 2003 ; Contini et al. 2005 ;
and multilocus sequence typing (MLST) of single- Calderaro et al. 2006 ; Edvinsson et al. 2006). The
copy DNA sequences belong to this group. In the qPCR approach has gained popularity for not only
following sections, we discuss the above methods in detecting but also quantifying T. gondii in biological
detail. samples (Bell and Ranford-Cartwright, 2002; Contini

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 Molecular detection and identification of Toxoplasma 3

et al. 2005). Its sensitivity is superior to n-PCR as- per replication (Goldstein and Schlotterer, 1999).
says (Contini et al. 2005) and can detect T. gondii Although the exact mutation rates of SNPs and
over a range of 6–7 orders of magnitude (Lin et al. microsatellites are not known in T. gondii, it is clear
2000 ; Jauregui et al. 2001 ; Contini et al. 2005). that the latter have a higher mutation rate and can
Quantification of T. gondii by qPCR has been ap- provide enhanced resolution in typing (Ajzenberg
plied to the detection of the parasite in animal tissues et al. 2002 a). The microsatellites are useful in
and whole blood or amniotic fluids of human clinical distinguishing genetically closely related T. gondii
samples (Lin et al. 2000 ; Jauregui et al. 2001 ; isolates. Since multilocus PCR-RFLP and micro-
Kupferschmidt et al. 2001 ; Contini et al. 2005). satellite typing are simple and cost effective com-
Since it can estimate the intensity of T. gondii pared with MLST typing, they are preferred tools
infection in the patients following drug treatment, for characterization of T. gondii in epidemiological
qPCR is extremely useful to monitor the progression studies.
of infections under treatment and to diagnose low- A hallmark study of the epidemiology and popu-
level infections or carrier states (Contini et al. 2005). lation structure of T. gondii was conducted more
The 529 bp element is a preferred target for qPCR, than a decade ago on 106 isolates collected from
as it provides a sensitivity of 6 repeat equivalents, or humans and animals in North America and Europe
1/50 of a genome equivalent (Kasper et al. 2009), (Howe and Sibley, 1995). In this study, 6 PCR-
currently representing the most sensitive assay for RFLP markers were used and 3 predominant lin-
the detection of T. gondii. eages (types I, II and III) were identified. It was
Rapid and accurate detection of T. gondii infection concluded that T. gondii had a clonal population
is pivotal for prompt treatment of clinical toxo- structure (Howe and Sibley, 1995). Since then,
plasmosis. However, the above-mentioned detection different sets of multilocus PCR-RFLP or micro-
methods (conventional PCR, n-PCR and qPCR of satellite markers have been applied to epidemi-
repetitive DNA sequences) provide no information ological and population studies (Blackston et al. 2001 ;
beyond a positive diagnosis. To better understand Ajzenberg et al. 2002 a, b, 2004, 2005 ; Lehmann et al.
the epidemiology of the parasite, there is an in- 2004, 2006 ; Khan et al. 2005 a, b; Ferreira et al.
creasing interest and need in the genetic classifi- 2006, 2008 ; Su et al. 2006). A key finding of these
cation of T. gondii. In the following section, we studies was that T. gondii isolates from South
discuss the methods used for the genetic identifi- America were highly diverse and distinct from those
cation and characterization of this parasite. of North America and Europe. It was concluded that,
in South America, T. gondii has an epidemic popu-
lation structure (Ajzenberg et al. 2004 ; Lehmann
Molecular characterization by multilocus
et al. 2004, 2006 ; Pena et al. 2008). However, the
PCR-RFLP, microsatellite or multilocus
comparing of genotypes among different studies has
sequence typing (MLST) analysis
been difficult or impossible, as different markers
For epidemiological studies, it is important to ident- have been employed in different laboratories.
ify individual T. gondii isolates and to track the The disadvantage of using a single-copy gene or
source of contamination. The commonly used meth- genomic sequence is that the sensitivity is compro-
ods for these purposes are multilocus PCR-RFLP, mised compared with a highly repetitive sequence. A
microsatellite or MLST typing. The PCR-RFLP is technical challenge to multilocus typing is that often
based on the ability of restriction endonucleases to samples are limited both in volume and in DNA
recognize single nucleotide polymorphisms (SNPs), amount, particularly from tissues from clinical cases
digest PCR products and subsequently display dis- or the mouse bioassay. To alleviate this problem,
tinct DNA banding patterns on agarose gels by we have developed a multiplex multilocus nested
electrophoresis (Sibley and Boothroyd, 1992 ; Howe PCR-RFLP (Mn-PCR-RFLP) method employing
and Sibley, 1995). Microsatellite analysis is based on 10 genetic markers, including SAG1, SAG2, SAG3,
the DNA sequence length polymorphisms of short BTUB, GRA6, c22-8, c29-2, L358, PK1 and Apico
nucleotide tandem repeats. The tandem repeats in (Dubey et al. 2007 e). The sensitivity of this method
T. gondii are often simple, consisting of as few as 2 is estimated at 10 or more T. gondii genome equiv-
nucleotides (di-nucleotide repeats), and occur 2–20 alents, based on a previous study with 4 (SAG2,
times (Blackston et al. 2001 ; Ajzenberg et al. 2002 a, SAG3, BTUB and GRA6) of these 10 markers
2004). The MLST is based on DNA sequence (Khan et al. 2005 a). In contrast, the sensitivity
polymorphisms including the SNPs, insertion and of conventional PCR-RFLP is estimated at o100
deletion of nucleotides in the sequence. SNPs and T. gondii genome equivalents (unpublished data).
microsatellites are predicted to mutate at different In Mn-PCR-RFLP typing, all markers are pre-
evolutionary rates. For eukaryotes in general, the amplified by multiplex PCR using external primers
mutation rate for SNPs is estimated at 10x9–10x10 in a single reaction, and the pre-amplified PCR
per nucleotide position per replication, whereas the products are used as templates to amplify each
rate for microsatellites is 10x2–10x5 per locus individual marker by n-PCR (Fig. 1). The advantage

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 C. Su and others 4

Table 1. Comparison of the methods for molecular detection and characterization of Toxoplasma gondii

Sensitivity (no. of parasites/ Advantage and

Method per PCR reaction) limitation Suitable application References

qPCR of 0.02 to 1 genome Highly sensitive in Diagnosis (Edvinsson et al. 2006 ;

repetitive equivalent at 529-bp detection. Isolation Kasper et al. 2009 ;
sequences locus. of parasite not needed. Reischl et al. 2003)
y1 genome equivalent Not suitable for typing. (Calderaro et al. 2006 ;
at B1 locus Costa et al. 2000 ;
Reischl et al. 2003)
n-PCR of 1 to 5 genome equivalent Sensitive. Isolation of Diagnosis (Calderaro et al. 2006 ;
repetitive at B1 locus parasite not needed. Contini et al. 2002)
sequences Not suitable for typing.
Mn-PCR-RFLP y10 genome equivalent Less sensitive. High Epidemiology and (Khan et al. 2005 a)
resolution in typing. population genetics
Isolation of parasite
may not be needed.
Mn-PCR-based y10 genome equivalent Less sensitive, high Epidemiology, This study
sequencing resolution in typing. population genetics
Isolation of parasite and phylogenetics
may not be needed.
Conventional 100 genome equivalent Insensitive, high Epidemiology, This study
PCR-based resolution in typing. population genetics
sequencing Isolation of parasite and phylogenetics
needed.
Multiplex PCR Not known High resolution in Epidemiology, (Ajzenberg et al. 2005)
of microsatellite typing population genetics
markers

B
Multiplex PCR using
external primers C

A’ B’ C’ D’

Multiplex PCR
products

Nested PCR of
individual locus
using nested
primers

Nested PCR products

RFLP genotyping Locus A’ Locus B’ Locus C’ Locus D’

Fig. 1. Multiplex multilocus nested PCR-RFLP (Mn-PCR-RFLP) typing. Several genetic loci are pre-amplified by
multiplex PCR with external primers. Then each individual locus is amplified by nested PCR using internal primers.
The nested PCR products are treated with restriction enzymes and resolved in agarose gel to reveal strain-specific DNA
banding patterns (RFLP typing). Horizontal lines represent DNA sequences and the horizontal arrows represent PCR
primers.

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 Table 2. Summary of primers for multiplex multilocus nested PCR-RFLP (Mn-PCR-RFLP) typing (Su and Dubey, 2009)

Molecular detection and identification of Toxoplasma

(The multiplex PCR reaction is carried out in a vol. of 25 ml containing 1r PCR buffer, 2 mM MgCl2, 200 mM each of the dNTPs, 0.15 mM each of the external forward and reverse
primers, 1 unit of FastStart DNA polymerase (Roche, Indianapolis, IN) and 1.5 ml of DNA samples. The reaction mixture is treated at 95 xC for 4 min, followed by 30 cycles of 94 xC
for 30 sec, 55 xC for 1 min and 72 xC for 2 min. Multiplex PCR amplified products are diluted (1 : 1) by adding 25 ml of nuclease-free water. The nested PCR reaction is carried out in
a vol. of 25 ml containing 1r PCR buffer, 2 mM MgCl2, 200 mM each of the dNTPs, 0.30 mM each of internal forward and reverse primers, 1 unit FastStart DNA polymerase and
1.5 ml of diluted multiplex PCR products. The reaction mixture is treated at 95 xC for 4 min, followed by 35 cycles of 94 xC for 30 sec, 60 xC for 1 min and 72 xC for 1.5 min (for
marker Apico, the annealing temperature is at 58 xC instead of 60 xC). For RFLP typing, 3–5 ml of nested PCR products are treated with restriction enzymes in a vol. of 20 ml and the
digested samples are resolved in agarose gel to reveal DNA banding patterns (Su et al. 2006).)

Nested
PCR Restriction enzymes, NEB buffers,
Markers Multiplex PCR primers (external primers)* Nested PCR primers (internal primers) (bp) incubation temperature and time Reference

SAG1 F : GTTCTAACCACGCACCCTGAG F: CAATGTGCACCTGTAGGAAGC 390 Sau96I+HaeII (double digest). (Grigg et al. 2001)
R : AAGAGTGGGAGGCTCTGTGA R: GTGGTTCTCCGTCGGTGTGAG NEB4, BSA, 37 xC 1 h. 2.5% gel.
5k-SAG2 Not needed. F: GAAATGTTTCAGGTTGCTGC 242 MboI, NEB4, BSA, 37 xC 1 h. (Howe et al. 1997 ;
The DNA fragment for 5k-SAG2 is covered R: GCAAGAGCGAACTTGAACAC 2.5% gel. Su et al. 2006)
by the external primers of alt. SAG2.
3k-SAG2 F : TCTGTTCTCCGAAGTGACTCC F: ATTCTCATGCCTCCGCTTC 222 HhaI, NEB4, BSA, 37 xC 1 h. (Howe et al. 1997)
R : TCAAAGCGTGCATTATCGC R: AACGTTTCACGAAGGCACAC 2.5% gel.
alt. SAG2 F : GGAACGCGAACAATGAGTTT F: ACCCATCTGCGAAGAAAACG 546 HinfI+TaqI, NEB3, BSA, 37 xC (Khan et al. 2005 b ;
R : GCACTGTTGTCCAGGGTTTT R: ATTTCGACCAGCGGGAGCAC 30 min, 65 xC 30 min. 2.5% gel. Su et al. 2006)
SAG3 F : CAACTCTCACCATTCCACCC F: TCTTGTCGGGTGTTCACTCA 225 NciI, NEB4, BSA, 37 xC 1 h. (Grigg et al. 2001)
R : GCGCGTTGTTAGACAAGACA R: CACAAGGAGACCGAGAAGGA 2.5% gel.
BTUB F : TCCAAAATGAGAGAAATCGT F: GAGGTCATCTCGGACGAACA 411 BsiEI+TaqI (double digest), (Khan et al. 2005 b ;
R : AAATTGAAATGACGGAAGAA R: TTGTAGGAACACCCGGACGC NEB4, BSA, 60 xC 1 h. 2.5% gel. Su et al. 2006)
GRA6 F : ATTTGTGTTTCCGAGCAGGT F: TTTCCGAGCAGGTGACCT 344 MseI, NEB2, BSA, 37 xC 1 h. (Khan et al. 2005 b ;
R : GCACCTTCGCTTGTGGTT R: TCGCCGAAGAGTTGACATAG 2.5% gel. Su et al. 2006)
C22-8 F : TGATGCATCCATGCGTTTAT F: TCTCTCTACGTGGACGCC 521 BsmAI+MboII (double digest), (Khan et al. 2005 b ;
R : CCTCCACTTCTTCGGTCTCA R:AGGTGCTTGGATATTCGC NEB2, BSA, 37 xC 30 min, 55 xC Su et al. 2006)
30min. 2.5% gel.
C29-2 F : ACCCACTGAGCGAAAAGAAA F: AGTTCTGCAGAGTGTCGC 446 HpyCH4IV+RsaI (double digest), (Khan et al. 2005 b ;
R : AGGGTCTCTTGCGCATACAT R:TGTCTAGGAAAGAGGCGC NEB1, BSA, 37 xC 1 h. 2.5% gel. Su et al. 2006)
L358 F : TCTCTCGACTTCGCCTCTTC F: AGGAGGCGTAGCGCAAGT 418 HaeIII+NlaIII (double digest), (Khan et al. 2005 b ;
R : GCAATTTCCTCGAAGACAGG R: CCCTCTGGCTGCAGTGCT NEB4, BSA, 37 xC 1 h. 2.5% gel. Su et al. 2006)
PK1 F : GAAAGCTGTCCACCCTGAAA F: CGCAAAGGGAGACAATCAGT 903 AvaI+RsaI (double digest), NEB4, (Khan et al. 2005 b ;
R : AGAAAGCTCCGTGCAGTGAT R: TCATCGCTGAATCTCATTGC BSA, 37 xC 1 h. 2.5% gel. Su et al. 2006)
Apico F : TGGTTTTAACCCTAGATTGTGG F: GCAAATTCTTGAATTCTCAGTT 640 AflII+DdeI (double digest), NEB2, (Su et al. 2006)
R : AAACGGAATTAATGAGATTTGAA R: GGGATTCGAACCCTTGATA BSA, 37 xC 1 h. 3% gel.

* F, forward primer ; R, reverse primer.

5
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 C. Su and others 6

TOWARD AN INTEGRATED APPROACH FOR

i
ond
T. gondii

um
MOLECULAR DETECTION AND IDENTIFICATION

ona
m

anin

eur
am
Each of the detection and identification methods

CTG
PGT
GT1

H. h

S. n
N. c
MK MK discussed above has its advantages and/or limi-
tations, depending on application. A comparison of
766 these methods is presented in Table 1. At present,
500
350 most clinical studies have focused on detecting the
250, 300 presence of T. gondii DNA in biological samples
200
150
using the highly sensitive nested or qPCR of repeti-
tive DNA sequences, but little has been done to
100
further characterize the parasites. This is because
75
each of these markers (B1 gene, 529 bp repetitive
50 element, or ITS-1) has identical or near-identical
repetitive sequences, therefore they are not adequate
25 for genetic typing. The conventional multilocus
PCR-RFLP analysis method relies on single-copy
polymorphic DNA sequences, and usually a rela-
Fig. 2. PCR-RFLP gel image for Toxoplasma gondii, tively large amount of starting templates from the
Hammondia hammondi, Neospora caninum and parasite is required. Therefore, it has been difficult
Sarcocystis neurona based on the 18S rDNA marker. to genetically identify T. gondii in clinical samples,
PCR products from each parasite species were
due to the extremely small amounts of parasite
digested with 3 restriction enzymes DdeI, MspI,
DNA available. The recent development of the
Hpy188III (New England Biolabs Inc.) in a single
reaction and DNA fragments were separated in 3% Mn-PCR-RFLP method makes it possible to gen-
agarose gels. Samples GT1, PTG and CTG represent etically characterize or classify T. gondii from clinical
T. gondii. All 4 apicomplexan species have unique samples with high resolution.
banding patterns and can be clearly distinguished Here, we propose an integrated approach for the
(mouse and human DNA were used as negative controls detection and genetic characterization of T. gondii.
and no PCR products were amplified, not shown). This approach should include detection of T. gondii
MK : Low molecular weight DNA ladder (New England infection in biological samples by n-PCR or qPCR
Biolabs Inc.). of repetitive DNA sequences (employing the B1
gene or the 529-bp repeat element), followed by
Mn-PCR-RFLP for the subsequent identification
of this approach is that only a limited amount of of T. gondii. There are a number of protocols de-
individual sample is needed, which is particularly veloped in several laboratories for the detection of
useful when only small amounts of ‘ precious ’ sam- T. gondii by qPCR or n-PCR (Costa et al. 2000 ;
ples are available. Mn-PCR-RFLP analysis has been Reischl et al. 2003 ; Edvinsson et al. 2006 ; Calderaro
applied to the genetic typing of a large number of et al. 2006 ; Kasper et al. 2009). Although the sen-
animal isolates bioassayed in mice, generating a sitivity of these protocols varies among different
substantial amount of data regarding genetic diver- laboratories, all studies demonstrate that the 529 bp
sity and population structure of the parasite (Dubey repeat element is the most sensitive target, with a
et al. 2007 a-g, 2008 a-e ; Pena et al. 2008 ; Sundar detection limit of o1/50 of a genome equivalent
et al. 2008 ; Velmurugan et al. 2008 ; Dubey and Su, (Kasper et al. 2009). Regardless of the protocol
2009). This method has also been used for the gen- employed, all test-positive samples should be further
etic characterization of T. gondii DNA extracted characterized genetically by the Mn-PCR-RFLP
from the brains of sea otters (Sundar et al. 2008) and typing method. The PCR primer sequences and
arctic foxes (Prestrud et al. 2008). Application of this experimental conditions for this typing method are
method to a broad range of samples will greatly listed in Table 2. This integrated approach will
facilitate our understanding of molecular epidemi- provide maximum information for epidemiological
ology and population diversity of T. gondii in the studies in order to address questions regarding the
near future. When the amount of DNA for T. gondii association of disease manifestations with parasite
isolates is not limited, the MLST typing is the genotypes and virulence.
method of choice because it has the highest res- For phylogenetic studies, MLST is preferred for
olution among all typing methods. Recent studies its high resolution. Conventional PCR based MLST
of T. gondii using this approach have revealed alleles requires the samples to have a relatively high con-
unique to parasite isolates in Brazil (Khan et al. centration of genomic DNA (o100 genome equiv-
2006, 2007), emphasizing the importance of a se- alents per PCR). Taking the Mn-PCR approach, we
quencing approach for studying the population have been able to generate quality DNA sequence
genetics and molecular phylogeny of T. gondii. data from samples containing as low as 10 genome

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 Molecular detection and identification of Toxoplasma 7

Table 3. Summary of multilocus PCR-RFLP typing for Toxoplasma gondii reference isolates

Genetic markers

T. gondii (5k+3k) alt.

reference isolates SAG1* SAG2# SAG2$ SAG3 BTUB GRA6 C22-8 C29-2 L358 PK1 Apico

GT1, RH88 (type I) I I I I I I I I I I I

PTG (type II) II or III II II II II II II II II II II
CTG (type III) II or III III III III III III III III III III III
TgCgCa1 (cougar, I II II III II II II u-1 I u-2 I
COUG)
MAS u-1 I II III III III u-1 I I III I
TgCatBr5 I III III III III III I I I u-1 I
TgCatBr64 I I u-1 III III III u-1 I III III I
TgRsCr1 u-1 I II III I III u-2 I I III I

* At SAG1 locus, type II and III are indistinguishable.

# SAG2 marker based on 5k- and 3k-ends of the gene sequence (Howe et al. 1997).
$ A SAG2 marker based on the 5k-end of the gene sequence but different from 5k-SAG2 (Su et al. 2006).
u-1 and u-2 are new alleles that are different from the clonal type I, II and III alleles.

SAG1 5’-SAG2 3’-SAG2 alt. SAG2

r64

r64
r64

r64
MA a1

r5

MA a1
MA a1

r5
r5

MA a1

r5
1

1
1

1
sC r
sCr
atB

sCr
atB
atB
atB

sC r

atB
atB

atB
gC

atB

gC
gC

gC
S

S
S

S
PGT

PGT
PGT
CTG

CTG
CTG

PGT
C TG
TgC

TgR

TgC
GT1

TgC
TgC

TgR
GT1

TgC
GT1

TgC
TgC

TgR

TgC

TgC
TgC

GT1

TgC
TgR
TgC
MK MK MK MK MK MK MK MK

766 766
500 500
350 350
250,300 250,300
200 200
150 150
100 100
75 75
50 50

25 I II III I u-1 I I u-1 III III I II III II I III I I I II III II II III u-1 II 25

SAG3 BTUB GRA6 c22-8

r64
r64

r64
r64

MA a1

r5
MA a1
MA a1

MA a1
r5

r5

r5

1
1

1
1

sCr
atB
sCr

sCr
sCr

atB

atB
atB

atB
atB

atB

atB

gC
gC
gC

gC

S
S

S
S

P GT
C TG
P GT
P GT

P GT

C TG
C TG

C TG

TgC

TgR
GT1

TgC
TgC
TgC

TgC

TgR

TgR
TgR

TgC

GT1

TgC
GT1

TgC

GT1

TgC
TgC

TgC

TgC

MK MK MK MK MK MK MK MK

766
766 500
500 350
350 250,300
250,300 200
200 150
150
100 100
75 75
50 50

I II III II III III III I I II III II III III III III I II III II u-1 I u-1 u-2 25
25 I II III III III III III III

c29-2 L358 PK1 Apico

r64
r64

r64
r64

MA a1

MA a1

MA a1

r5
r5

r5
MA a1

r5

1
1

sCr

sCr

sCr
atB
atB

atB
sCr
atB

atB
atB

atB
atB

gC

gC

gC
gC

S
S

P GT

PGT

PGT
C TG
P GT

CTG

CTG
C TG

TgC

TgC

TgC
TgR

TgR

TgR
GT1

TgC
TgC

TgC
GT1

GT1
TgC

TgR
GT1

TgC

TgC
TgC

TgC
TgC

MK MK MK MK MK MK MK MK

766 766
500 500
350
350 250,300
250,300 200
200 150
150 100
100 75
75
50
50

25 I II III u-1 I I I I I II III I I I III I I II III u-2 III u-1 III III I II III I I I I I 25

Fig. 3. Multiplex multilocus nested PCR-RFLP (Mn-PCR-RFLP) analysis of Toxoplasma gondii samples using
10 different genetic markers. T. gondii reference strains are GT1 (or RH88), PTG, CTG, TgCgCa1 (a.k.a. Cougar,
COUG), MAS, TgCatBr5, TgCatBr64 and TgRsCr1. Nested PCR products from each marker were digested with
selected restriction enzymes (Table 2), and DNA fragments were separated in agarose. All markers were resolved in
2.5 % gels, except that Apico was resolved in 3% gels. MK, low molecular weight DNA ladder (New England Biolabs
Inc.).

equivalent (data not published). The major pre- generated. This problem can be solved by sequenc-
caution for this approach is that, if errors occur in ing at least 2 independent PCR products from the
the early cycles of PCR, erroneous sequences may be same sample. To standardize studies and facilitate

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 C. Su and others 8

comparison among different laboratories, it is strongly were designated either as I, II, III, or 1, 2, 3, re-
recommended that all sequencing efforts include spectively (Darde et al. 1992 ; Sibley and Boothroyd,
a few commonly used introns such as UPRT-1, 1992 ; Ajzenberg et al. 2004 ; Ferreira et al. 2006 ;
UPRT-7, EF1 and HP (Khan et al. 2006, 2007) and Khan et al. 2006 ; Su et al. 2006 ; Pena et al. 2008).
the GRA6 gene (Fazaeli et al. 2000). Alleles which are distinct from those of types I, II
For genomic DNA samples extracted directly or III were designated as either unique-1 (u-1),
from animal tissues, we suggest screening all sam- unique-2 (u-2), unique-3 (u-3) etc., or 4, 5, 6, etc. In
ples using n-PCR targeting the 18S rDNA sequence Table 3 and Fig. 3, we use the former scheme to
prior to Mn-PCR-RFLP typing for T. gondii. By describe new alleles.
sequence analysis of the 18S rDNA for a number of In summary, the application of an integrated
closely related apicomplexan parasites, we identified approach, combining molecular detection and high
SNPs that can distinguish T. gondii, Hammondia resolution genetic characterization, should assist in
hammondi, Neospora caninum and Sarcocystis neurona enhancing our understanding of the molecular epi-
by RFLP analysis (Fig. 2). For n-PCR of 18S demiology, population genetics and phylogeny of
rDNA, the external primers are : Tg18s48F, CCA T. gondii, which will be beneficial to the control of
TGCATGTCTAAGTATAAGC and Tg18s359R, T. gondii transmission and a reduction of toxo-
GTTACCCGTCACTGCCAC, which amplify a plasmosis in humans and animals.
312bp DNA fragment from T. gondii. The nested
PCR primers are : Tg18s58F, CTAAGTATAAGC
ACKNOWLEDGEMENTS
TTTTATACGGC and Tg18s348R, TGCCACG
GTAGTCCAATAC, which amplify a 291bp DNA This work was supported in part by the Department of
fragment from T. gondii. These primers were de- Microbiology, University of Tennessee Knoxville (CS,
Startup grant) ; the Chinese National Special Research
signed based on sequences conserved for the above Program for Non-Profit Trades (XQZ, Grant No.
4 parasites. The GenBank Accession numbers of 200803017) ; and the Program for Changjiang Scholars and
these sequences are M97703 (T. gondii), AF096498 Innovative Research Team in University – China (XQZ,
(H. hammondi), U17346 (N. caninum) and U07812 Grant No. IRT0723).
(S. neurona). Using this marker, we recently de-
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