Review

Advances in molecular identication, taxonomy, genetic variation and diagnosis

of Toxocara spp.
Jia Chen
a,b,1
, Dong-Hui Zhou
a,1
, Alasdair J. Nisbet
c
, Min-Jun Xu
a
, Si-Yang Huang
a
, Ming-Wei Li
a,d
,
Chun-Ren Wang
e
, Xing-Quan Zhu
a,e,f,
a
State Key Laboratory of Veterinary Etiological Biology, Key Laboratory of Veterinary Parasitology of Gansu Province, Lanzhou Veterinary Research Institute, Chinese Academy
of Agricultural Sciences, Lanzhou, Gansu Province 730046, PR China
b
College of Veterinary Medicine, South China Agricultural University, Guangzhou, Guangdong Province 510642, PR China
c
Parasitology Division, Moredun Research Institute, Pentlands Science Park, Midlothian EH26 0PZ, Scotland, UK
d
Department of Veterinary Medicine, Agricultural College, Guangdong Ocean University, Huguangyan, Zhanjiang, Guangdong Province 524088, PR China
e
College of Animal Science and Veterinary Medicine, Heilongjiang Bayi Agricultural University, Daqing, Heilongjiang Province 163319, PR China
f
College of Animal Science and Technology, Yunnan Agricultural University, Kunming, Yunnan Province 650201, PR China
a r t i c l e i n f o
Article history:
Received 5 February 2012
Received in revised form 20 April 2012
Accepted 21 April 2012
Available online 28 April 2012
Keywords:
Toxocara
Toxocariasis
Molecular identication
Molecular taxonomy
Genetic variation
Molecular diagnosis
a b s t r a c t
The genus Toxocara contains parasitic nematodes of human and animal health signicance, such as Tox-
ocara canis, Toxocara cati and Toxocara vitulorum. T. canis and T. cati are among the most prevalent para-
sites of dogs and cats with a worldwide distribution. Human infection with T. canis and T. cati, which can
cause a number of clinical manifestations such as visceral larva migrans (VLMs), ocular larva migrans
(OLMs), eosinophilic meningoencephalitis (EME), covert toxocariasis (CT) and neurotoxocariasis, is con-
sidered the most prevalent neglected helminthiasis in industrialized countries. The accurate identica-
tion Toxocara spp. and their unequivocal differentiation from each other and from other ascaridoid
nematodes causing VLMs and OLMs has important implications for studying their taxonomy, epidemiol-
ogy, population genetics, diagnosis and control. Due to the limitations of traditional (morphological)
approaches for identication and diagnosis of Toxocara spp., PCR-based techniques utilizing a range of
genetic markers in the nuclear and mitochondrial genomes have been developed as useful alternative
approaches because of their high sensitivity, specicity, rapidity and utility. In this article, we summarize
the current state of knowledge and advances in molecular identication, taxonomy, genetic variation and
diagnosis of Toxocara spp. with prospects for further studies.
2012 Elsevier B.V. All rights reserved.
Contents
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1344
2. Molecular identification, taxonomy and genetic variation of Toxocara spp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1345
3. Molecular detection and diagnosis of Toxocara spp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1346
4. Future prospects. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1347
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1347
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1347
1. Introduction
Toxocara is an important ascaridoid genus containing species of
human and animal health signicance, such as Toxocara canis, Tox-
ocara cati and Toxocara vitulorum (Magnaval et al., 2001; Despom-
mier, 2003; Lee et al., 2010; Rubinsky-Elefant et al., 2010). T. canis
and T. cati are among the most prevalent endoparasites in their
denitive hosts (dogs and cats), having a worldwide distribution
(Magnaval et al., 2001). Epidemiological surveys have indicated
1567-1348/$ - see front matter 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.meegid.2012.04.019

Corresponding author at: State Key Laboratory of Veterinary Etiological Biology,

Key Laboratory of Veterinary Parasitology of Gansu Province, Lanzhou Veterinary
Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, Gansu
Province 730046, PR China. Tel.: +86 931 8342837; fax: +86 931 8340977.
E-mail address: xingquanzhu1@hotmail.com (X.-Q. Zhu).
1
These authors contributed equally to this work.
Infection, Genetics and Evolution 12 (2012) 13441348
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that the prevalence of T. canis in dogs ranged from 5.5% to 64.7%
(Minnaar et al., 2002; Oliveira-Sequeira et al., 2002; Habluetzel
et al., 2003; Rubel et al., 2003; Wang et al., 2006; Dai et al.,
2009), and the prevalence of T. cati in cats ranged from 4.7% to
55.2% (Calvete et al., 1998; Martnez-Barbabosa et al., 2003; Mir-
cean et al., 2010; Barutzki and Schaper, 2011; Khalafalla, 2011).
Widespread prevalence of Toxocara spp. in dogs and cats has led
to the contamination of playgrounds, municipal parks and house-
holds with Toxocara eggs (Deplazes et al., 2011; Dado et al.,
2012; Mattia et al., 2012).
Signicantly, humans can be infected by T. canis and T. cati
through accidental ingestion of embryonated eggs. Although the
larval stages are unable to develop to mature adult worms in hu-
mans, infective Toxocara larvae may migrate to a range of tissues,
causing damage to whatever tissue they happen to enter, resulting
in a number of clinical manifestations such as visceral larva mi-
grans (VLMs), ocular larva migrans (OLMs), eosinophilic meningo-
encephalitis (EME), covert toxocariasis (CT) and neurotoxocariasis
(Despommier, 2003; Fisher, 2003; Vidal et al., 2003; Finsterer
and Auer, 2007; Smith et al., 2009; Lee et al., 2010; Rubinsky-Ele-
fant et al., 2010). Recent epidemiological data demonstrate the
widespread prevalence of human infection with Toxocara, which
is probably the most prevalent helminthiasis in industrialized
countries, representing a typical neglected and underestimated hu-
man health problem (Magnaval et al., 2001; Despommier, 2003;
Lee et al., 2010; Rubinsky-Elefant et al., 2010; Carvalho and Rocha,
2011; Turrientes et al., 2011).
Owing to their human and animal health signicance, a large
number of studies have been performed on many aspects of Toxo-
cara spp., such as taxonomy, epidemiology, pathogenesis, diagnosis
and treatment as well as molecular aspects relating to their identi-
cation, genetic variation and diagnosis (Magnaval et al., 2001;
Zhu et al., 2001; Despommier, 2003; Gasser et al., 2006; Lee
et al., 2010; Rubinsky-Elefant et al., 2010). This article reviews
the current state of knowledge, advances in molecular identica-
tion, taxonomy, genetic variation and diagnosis of Toxocara spp.
and highlights prospects for further studies.
2. Molecular identication, taxonomy and genetic variation of
Toxocara spp.
Traditionally, ascaridoid nematodes of the genus Toxocara have
been identied and classied based on their morphological fea-
tures and predilection sites in a particular host species (Skrjabin
et al., 1991; Bowman, 2009; Borecka et al., 2010). A number of Tox-
ocara species have been described, such as T. canis (from canids), T.
cati (from felines), Toxocara lyncus (from caracals), T. vitulorum
(from bovids), Toxocara tanuki (from canids), Toxocara apodemi
and Toxocara mackerrasae (from rodents), Toxocara paradoxura
and Toxocara sprenti (from viverrids), Toxocara vajrasthirae (from
mustelids) and Toxocara pteropodis (from bats) (Skrjabin et al.,
1991; Borecka et al., 2010). However, there can be considerable
limitations in traditional methods for the accurate identication,
differentiation and taxonomy of some Toxocara species, especially
at the larval and/or egg stages, which has raised questions in the
taxonomic analyses of Toxocara (Zhu et al., 1998, 2001; Gasser
et al., 2006).
Utilizing sequences of the second internal transcribed spacer
(ITS-2) of nuclear ribosomal DNA (rDNA), Jacobs et al. (1997) were
the rst group to demonstrate that morphologically well-dened
adults of T. canis, T. cati and Toxascaris leonina from dogs and cats
could be distinguished by their respective ITS-2 sequences, and
the three species could be differentiated from each other and from
other ascaridoids that may be found in human tissues by direct PCR
and PCR-linked restriction fragment length polymorphism (PCR-
RFLP) assays based on ITS-2 sequences.
In a further study, the sequences of the rst internal transcribed
spacer (ITS-1) and the ITS-2 sequences were used to characterize
an ascaridoid nematode of cats from Kuala Lumpur, Malaysia, pre-
viously identied morphologically as T. canis (Lee et al., 1993).
Comparative analysis of the ITS-1 and ITS-2 sequences revealed
the existence of a previously unknown Toxocara species, initially
designated Toxocara spp. cf. canis (Zhu et al., 1998), which is nei-
ther T. canis nor T. cati but represents a distinct species. Detailed
morphological study of this Toxocara variant from Malaysia sup-
ported this conclusion, and the parasite was described and named
as Toxocara malaysiensis (Gibbons et al., 2001). T. malaysiensis was
shown to be present in cats in Guangzhou, China by molecular
characterization using ITS-2 sequences as genetic markers (Li
et al., 2006). This study demonstrated the usefulness of ITS rDNA
sequences and molecular approaches (utilizing these genetic
markers) in the identication and characterization of morphologi-
cally very closely related, or cryptic, species (Nadler and DE Len,
2011).
Extending from these studies, Li et al. (2007) established spe-
cic PCR assays for the unequivocal identication and differentia-
tion of T. canis, T. cati, T. malaysiensis and Ta. leonina of dogs and
cats using species-specic primers designed using their ITS-1 and
ITS-2 sequences. These specic PCR assays were both sensitive
and specic, and provide molecular tools for diagnosis and for
molecular epidemiological surveys of Toxocara infections in hu-
mans and animals.
In addition to nuclear ITS-1 and ITS-2 rDNA sequences, recent
studies have shown that sequences derived from the mitochondrial
(mt) genome provide useful alternative genetic markers for inves-
tigating population genetic structures, systematics and phylogeny
of parasitic nematodes due to their higher mutation rates than nu-
clear genes (Hu et al., 2004; Hu and Gasser, 2006). Complete mt
genomic sequences have been determined for T. canis, T. cati, T.
malaysiensis (Li et al., 2008a) and nearly two-thirds (10,486 bp)
of the mt genome has been sequenced for T. vitulorum (Wickrama-
singhe et al., 2009), providing novel mitochondrial DNA (mtDNA)
markers for taxonomic and phylogenetic relationship analyses of
Toxocara species. Reconstruction of phylogenetic relationships
among Toxocara species using concatenated amino acid sequences
of 12 mitochondrial protein-coding genes indicated that T. malaysi-
ensis (from cats) was phylogenetically more closely related to T.
cati (from cats) than to T. canis (from dogs) (Li et al., 2008a), which
is consistent with results described by Zhu et al. (1998) using ITS-1
and ITS-2 sequences as genetic markers. Based on the 10,486 bp
mtDNA sequences (including protein-coding, intergenic, trn and
non-coding sequence), T. vitulorum was phylogenetically more clo-
sely related to T. malaysiensis than to T. canis and T. cati (Wickrama-
singhe et al., 2009), which raised concerns regarding its zoonotic
potential.
Previous studies have demonstrated the widespread existence
of genetic variation among parasite populations, and many have
focused on the accurate analysis of this variation with regard to
systematics, population structure and epidemiology of parasites,
and the application of this information for the effective control of
parasitic diseases (Zhu et al., 2001; Gasser, 2006).
Zhu et al. (1998) reported genetic variation in T. cati by demon-
strating that the ITS-1 sequence of T. cati from Malaysia was 9 bp
shorter than that derived from the Australian isolate. In addition
the ITS-1 nucleotide sequences derived from the two isolates dif-
fered by 2.9%. A single point mutation was found in the ITS-2 se-
quence between T. cati samples from Malaysia and Australia (Zhu
et al., 1998).
Employing single strand conrmation polymorphism (SSCP)
analysis (Gasser, 2006) combined with sequencing of ITS-2 PCR
J. Chen et al. / Infection, Genetics and Evolution 12 (2012) 13441348 1345
products, sequence microheterogeneity in the ITS-2 rDNA was
demonstrated within and/or among individual nematodes of T. cati
from Australia, and four different ITS-2 sequence types were iden-
tied, reecting genetic variation among different populations of T.
cati (Zhu and Gasser, 1998). Using partial sequences of mitochon-
drial cytochrome c oxidase subunit 1 (pcox1), NADH dehydroge-
nase subunits 1 and 4 (pnad1 and pnad4) as markers, genetic
variation among and within T. canis, T. cati, T. malaysiensis, T. vitu-
lorum and Ta. leonina from different geographical origins was
examined by Li et al. (2008b) using an SSCP-sequencing approach.
This study demonstrated the existence of intra-specic sequence
variations within each of the ve ascaridoid species (0.23.7% for
pcox1, 02.8% for pnad1 and 02.3% for pnad4), and revealed signif-
icantly higher sequence differences between the ve ascaridoid
species (7.912.9% for pcox1, 10.721.1% for pnad1 and 12.9
21.7% for pnad4), indicating the signicance of using mtDNA se-
quences as genetic markers for the studies of genetic variability
and for identication of Toxocara spp.
3. Molecular detection and diagnosis of Toxocara spp.
With the increasing number of dogs and cats being kept as pets
worldwide, more T. canis and T. cati eggs are excreted into the envi-
ronment, resulting in the accumulation of Toxocara eggs in the soil
in public and private areas (Gawor et al., 2008; Borecka et al.,
2010). Recent investigations have indicated the widespread con-
tamination of urban public parks with Toxocara eggs in many coun-
tries (e.g., Paquet-Durand et al., 2007; Avcioglu and Burgu, 2008;
Borecka et al., 2010; Jarosz et al., 2010), which has contributed to
the increase of human infection with Toxocara (Giacometti et al.,
2000; Gawor and Borecka, 2004; Muradian et al., 2005; Borecka
et al., 2010). Hence, the accurate detection and surveillance of
environmental contamination with Toxocara eggs is important for
the effective prevention and control of human infection with Tox-
ocara. However, several studies have indicated the difculty in dis-
tinguishing between T. canis and T. cati eggs because of their
similar morphologies and sizes (Fogt-Wyrwas et al., 2007; Borecka
and Gawor, 2008).
Due to the inherent limitations of traditional (morphological)
approaches, various DNA-based approaches, in particular PCR-
based techniques, have been developed and used for accurate
identication and diagnosis of Toxocara spp. because of their high
sensitivity, specicity, rapidity and utility (Zhu et al., 2001; Gasser
et al., 2006) (Table 1).
A previous study by Turcekova and Dubinsky (1996) indicated
the potential of restriction fragment length polymorphism (RFLP)
analysis of genomic DNA combined with DNA hybridization for
the identication and differentiation of T. canis and T. cati, but this
method requires the use of a large quantity of genomic DNA which
is not readily available for parasites of small sizes, particularly at
larval or egg stages.
PCR technology that permits specic amplication of minute
amounts of genomic DNA such as those from single nematode eggs
or worms can overcome the above limitations (reviewed by Gasser,
2006). For example, employing primers based on the sequence of a
particular fragment from random amplied polymorphic DNA pat-
tern (RAPD) analyses, Wu et al. (1997) developed a PCR assay for
the specic amplication of both T. canis and T. cati DNA samples.
This assay could not be used to distinguish between eggs of T. canis
and T. cati, and low reproducibility and specicity are the major
limitations of the RAPD technology (Gasser, 2006).
Because of the limitations of RFLP and RAPD techniques, a re-
cent study (Fogt-Wyrwas et al., 2007) established a specic PCR
protocol for the detection and differentiation of T. canis and T. cati
eggs isolated from soil samples utilizing the species-specic prim-
ers rst reported by Jacobs et al. (1997). In spite of its ability to de-
tect a single egg recovered from soil sample of 40 g, this method
has a limitation in that it requires the isolation of eggs from soil
samples by otation prior to the extraction of genomic DNA from
the isolated eggs, which is time consuming and labor intensive.
In order to overcome this limitation, Borecka and Gawor (2008)
modied the protocol by using proteinase K to digest the sand
(soil) samples prior to the extraction of genomic DNA, thus obtain-
ing Toxocara genomic DNA from a soil sample directly, without the
need for either the isolation of eggs by otation or the inactivation
of PCR inhibitors in soil samples. The subsequent PCR amplication
of the DNA templates using species-specic primers reported by
Jacobs et al. (1997) allowed the routine identication and differen-
tiation of T. canis eggs from T. cati eggs in soil samples contami-
nated with Toxocara eggs. This approach could be used for
studying the association between the prevalence of toxocariasis
among humans and the degree of soil contamination with Toxocara
eggs. However, these conventional specic PCR assays are usually
time consuming, involve many steps, and require the use of expen-
sive PCR thermocyclers.
In order to overcome these limitations of conventional specic
PCR assays, Notomi et al. (2000) developed a novel DNA amplica-
tion technique named loop-mediated isothermal amplication
(LAMP). This method does not require the use of normal PCR cy-
clers, instead, it requires only a water bath or heat block, can spe-
cically amplify target DNA into a large amount under isothermal
conditions in <1 h, and the amplication products can be detected
visually (Tomita et al., 2008). By adapting this technique, a recent
study (Macuhova et al., 2010) established a LAMP assay for the ra-
pid and reliable detection and discrimination of T. canis and T. cati
eggs in soil samples. Under laboratory conditions, this LAMP assay
could reliably detect as few as 3 T. canis eggs in articially contam-
inated sand. In a eld trial, this technique readily detected T. cati
DNA from natural sandpits with moderate or heavy contamination
with Toxocara eggs, offering a cheap, powerful and convenient ap-
proach for monitoring the contamination of soil with Toxocara
eggs.
Table 1
Summary of main molecular approaches used for identication and diagnosis of
Toxocara spp.
Method Main purposes DNA target
regions
Selected
references
RFLP Specic identication and
diagnosis of adults
Total
genomic
DNA
Turcekova
and
Dubinsky
(1996)
PCR-RAPD Amplication of eggs of T.
canis and T. cati, but
neither identication nor
detection
A particular
fragment of
genomic
DNA
Wu et al.
(1997)
Specic PCR for
soil sample
Specic identication and
differentiation of T. canis
and T. cati eggs in soil
indirectly
ITS-1; ITS-2 Fogt-
Wyrwas
et al. (2007)
Specic PCR
combined
with modied
DNA
extraction
Specic identication and
differentiation of T. canis
and T. cati eggs in soil
directly
ITS-1; ITS-2 Borecka and
Gawor
(2008)
LAMP Specic identication and
differentiation of T. canis
and T. cati eggs in soil
directly
ITS-2 Macuhova
et al. (2010)
Specic PCR for
tissue sample
Specic identication and
differentiation of Toxcara
larvae in tissues
ITS-1; ITS-2 Rai et al.
(1997),
Ishiwata
et al. (2004)
1346 J. Chen et al. / Infection, Genetics and Evolution 12 (2012) 13441348
It has been shown that infection of humans with infective lar-
vae of T. canis and T. cati can cause VLMs and OLMs with signicant
consequences (Despommier, 2003; Fisher, 2003; Gasser et al.,
2006). In addition to Toxocara spp., some other ascaridoid species
have also been shown to be involved in human infections (Smyth,
1995). For example, the larvae of the raccoon ascarid Baylisascaris
procyonis can invade humans and cause OLMs and VLMs (Smyth,
1995). Obviously the accurate identication and differentiation of
the causative agents is important for the proper treatment and
effective control of human VLMs and OLMs. Nevertheless, it has
been difcult, or even impossible, to distinguish between the lar-
vae of Toxocara spp. as well as larvae of other ascaridoid species
causing human VLMs and OLMs based on morphology because of
their morphological similarities, particularly when the larvae (or
parts of a larva) have been recovered from human tissues (Nichols,
1956; Gasser et al., 2006).
PCR-based approaches have therefore provided useful alterna-
tives for the accurate identication and differentiation of Toxocara
spp. larvae as well as larvae of other ascaridoids which cause VLMs
and OLMs. The ability of the reported specic PCR assays (e.g., Ja-
cobs et al., 1997; Li et al., 2007) to not amplify host (including hu-
man) DNA opened up the potential to detect and differentiate
Toxocara spp. larvae in human tissues directly, without the need
for localizing and isolating the larvae. An experimental study in
a mouse model (Rai et al., 1997) specically detected Toxocara lar-
vae in live biopsy materials by double PCR using Toxocara primers.
This method detected Toxocara spp. larvae without the need for
localizing and isolating the larvae, but was unable to distinguish
between species. Another study (Ishiwata et al., 2004) using PCR
amplication of ITS-2 rDNA combined with sequence analysis
identied the larva of T. canis embedded in a turkey liver. These
studies have indicated the possibility of using PCR-based technol-
ogy for the etiological diagnosis of VLMs and OLMs in humans. One
limitation of PCR-based diagnosis of human VLMs and OLMs is
that the time-consuming recovery of the larvae needs invasive
procedures.
4. Future prospects
This review has indicated signicant advances and achieve-
ments in molecular identication, taxonomy and diagnosis of Tox-
ocara spp., which has served as an example for tackling taxonomic
questions in other groups of parasites, such as Fasciola (e.g., Ai
et al., 2011), Orientobilharzia (Wang et al., 2011) and Trichuris
(Liu et al., 2012). However, there remain a number of areas requir-
ing further investigations of Toxocara spp.
To date, investigations of genetic variability among populations
of Toxocara spp. have focused on T. cati. Given the worldwide dis-
tribution of Toxocara spp., it is likely that unrecognized genetic
diversity may exist among populations of Toxocara spp. (reviewed
by Jenkins et al., 2011). A deeper understanding of genetic varia-
tion among populations of Toxocara spp. should therefore be
gained by utilizing more variable genetic markers and more sensi-
tive detection systems.
Exploiting the recent signicant advances and applications of a
range of omic (e.g., genomic, transcriptomic, proteomic, metabolo-
mic) technologies in parasites of major human and animal health
signicance (e.g., Gasser et al., 2011; Jex et al., 2011; Young
et al., 2012) will provide unique opportunities to address many
important fundamental areas of Toxocara, such as their genomes,
transcriptomes, proteomes and mircroRNAs, in a way which has
not been possible previously. These in-depth fundamental studies
will provide unprecedented resources for the development of
new intervention strategies for human and animal infections with
Toxocara spp.
Acknowledgements
Research in the authors laboratories is supported, in part, by
the Program for Outstanding Scientists in Agricultural Research,
the Open Funds of the State Key Laboratory of Veterinary Etiolog-
ical Biology, Lanzhou Veterinary Research Institute, Chinese Acad-
emy of Agricultural Sciences (Grant Nos. SKLVEB2011KFKT004,
SKLVEB2011KFKT011, SKLVEB2010KFKT009, SKLVEB2009KFKT008,
and SKLVEB2011KFKT010) and the Yunnan Provincial Program for
Introducing High-level Scientists (Grant No. 2009CI125).
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