Veterinary Parasitology 193 (2013) 337–341

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Veterinary Parasitology

jo urnal homepage: www.elsevier.com/locate/vetpar

Detection and identiﬁcation of Toxocara canis DNA in bronchoalveolar

lavage of infected mice using a novel real-time PCR

∗

E. Pinelli , J.H. Roelfsema, S. Brandes, T. Kortbeek

Centre for Infectious Disease Control Netherlands, National Institute of Public Health and the Environment, Postbus 1, Bilthoven, The Netherlands

a r t i c l e i n f o

a b s t r a c t

Keywords:

Toxocarosis is a zoonosis with worldwide distribution caused by Toxocara spp. of dogs

Toxocara canis

and cats. In humans, diagnosis relies mainly on detection of parasite-speciﬁc antibod-

Real-time PCR

ies. Although serological assays in current use have deﬁned sensitivity and speciﬁcity, the

Bronchoalveolar lavage

problem of cross-reactivity still remains, particularly in areas of endemic polyparasitism.

Ascaridoidea

Microscopic detection of the parasite in tissue biopsies is not recommended for diagnosis

Experimental infection

because larvae can be difﬁcult to locate, and ﬁnding the parasite eggs in faeces is not appli-

cable since the larvae do not develop to the adult stage in the human host. In this study

we describe a novel real-time PCR (‘Nemo-PCR’) that, in combination with DNA sequenc-

ing, allows the detection and identiﬁcation of Toxocara canis and other nematodes in the

Superfamily Ascaridoidea. Results indicate that this approach can detect Toxocara spp. DNA

in bronchoalveolar lavage (BAL) of experimentally-infected mice. For diagnostic purposes

further studies are necessary to evaluate this assay including testing human BAL ﬂuid. The

availability of such a direct assay would improve diagnosis of toxocarosis particularly for

1. Introduction

loss and hepato-splenomegaly. Other symptoms include

eosinophilic pneumonitis (Loefﬂer’s pneumonia) which

Toxocara canis and Toxocara cati are roundworms of dogs bear a clinical resemblance to the pulmonary inﬂamma-

and cats respectively and the causative agents of human tory responses observed in asthmatic patients (reviewed

toxocarosis. In their normal hosts, adult female worms in Smith et al., 2009).

shed large number of eggs in faeces contaminating sand- Diagnosis of human toxocarosis is based on serology in

pits, backyards and playgrounds. Humans become infected combination with the medical symptoms and signs of the

after ingesting embryonated eggs present in contaminated disease and laboratory ﬁndings such as eosinophilia, leuco-

soil. Once ingested, the larvae hatch, penetrate the intes- cytosis and hyperglobulinemia. Direct detection of larvae

tine and migrate via the blood vessels to different organs in tissues by microscopy from biopsies is rarely success-

including the lungs. In humans, larvae do not develop to ful. Furthermore, humans are accidental hosts and patent

the adult stage but can persist in tissues for many years infections do not result; therefore eggs are not produced,

(reviewed in Pinelli and Aranzamendi, 2012). Migration of making coprological techniques irrelevant. Serological

Toxocara spp. larvae through different organs may result assays have been validated (reviewed in Rubinsky-Elefant

in the syndrome known as Visceral Larva Migrans (VLM) et al., 2010; Smith et al., 2009) however, they are indi-

characterized by fever, cough, wheezing, malaise, weight rect assays and cross-reactivity with other helminths that

occur in different countries of the world cannot be excluded

(Lozano et al., 2004).

∗ In this study we describe a newly developed real-time

Corresponding author. Tel.: +31 30 2744277; fax: +31 30 2744418.

PCR for detection and identiﬁcation of nematodes from the

E-mail address: [email protected] (E. Pinelli).

338 E. Pinelli et al. / Veterinary Parasitology 193 (2013) 337–341

Table 1

Superfamily Ascaridoidea, referred to as the ‘Nemo-PCR’.

Sequence of forward (F) and reverse (R) primers and probe used in the

Importantly, this PCR was able to detect and identify T. canis

Nemo-real-time PCR.

DNA in bronchoalveolar lavage (BAL) of infected mice.

Primer-probe designation DNA sequence

Nemo 18S F ggctaagccatgcatgtc

Materials and methods

Nemo 18S R acttgatagacacgtcgcc a b c

Nemo TP1 6FAM -aaaccgcgaacggctcat-BHQ1

2.1. Experimental infection with T. canis a

Dual labeled Taqman probe; ﬂuorochrome.

6Fam stands for .6-carboxyﬂuorescein or, also known as 6-

T. canis eggs were collected from the uteri of female CarboxyFluorescein-Aminohexyl Amidite.

worms that were recovered from de-wormed, naturally Black Hole Quencher number 1.

infected dogs. The eggs were allowed to embryonate in

0.1 M H2SO4 at room temperature for 4–6 weeks. Embry- 2.4. Primer and probe design for detection of DNA from

◦

onated eggs were stored in 0.1 M H2SO4 at 4 C until use. nematodes from the Superfamily Ascaridoidea

Speciﬁed pathogen-free female BALB/c mice either

5–6 weeks or 1–2 weeks old were obtained from Har- Several sequences from the 18S rRNA gene from

lan Netherlands BV (Horst, The Netherlands). Mice were nematodes that belong to the Superfamily Ascaridoidea

housed in Macrolon III cages with food and water ad lib. including Anisakis spp., Ascaris spp. and Toxocara spp. were

Infection was performed by oral administration of 1000 aligned and suitable regions for primer and probe anneal-

embryonated eggs per mouse in 0.3 ml sterile phosphate- ing were determined using the software Primer and probe

buffered saline (PBS) using a syringe ﬁtted with a blunt design from Roche. A pair of primers and probe was cho-

needle. The uninfected mice received oral administration of sen and the Nemo-PCR was developed in order to amplify

0.3 ml PBS only. Four groups of mice with 6 mice per group a region that could, after sequencing, reveal which species

were studied: (a) 5–6 weeks old T. canis infected; (b) 5–6 from the Superfamily Ascaridoidea was ampliﬁed based on

weeks old uninfected; (c) 1–2 weeks old T. canis-infected the nucleotide composition. Table 1 shows the sequence of

and (d) 1–2 weeks old uninfected. Infected and uninfected the designed Nemo primers and probe (Biolegio, Nijmegen,

mice were euthanized at 60 days post-infection. All exper- The Netherlands) used.

iments were performed according to the International and

Institutional Guidelines for Animal Care. 2.5. Detection of T. canis DNA using a real-time PCR assay

␮

The Nemo-PCR consists of 4 l Taqman Mastermix

2.2. Larval counts

(Roche), 1.4 ␮l forward and reverse primers (10 pmol/␮l),

0.4 ␮l double labeled hydrolysis probe (5 pmol/␮l) and 5 ␮l

At 60 days post-infection, mice were euthanized and

DNA in a total volume of 20 ␮l. The reaction conditions

T. canis larvae were recovered from the lungs using the ◦

were: 10 min at 95 C, followed by 45 cycles of 10 s dena-

Baermann technique (Pritchard and Kruse, 1982) modi- ◦ ◦

turation at 95 C, 20 s annealing at 58 C and 20 s extension

ﬁed to reduce evaporation (Hamilton et al., 2006). Brieﬂy, ◦

at 72 C, using a Lightcycler 480 (Roche).

the macerated left or right lungs were added to a 15 ml

tube to which a double layer of gauze had been ﬁxed

2.6. Sequencing of PCR products

to the open-ended bottom. The 15 ml tube was placed

into a conical 50 ml centrifuge tube, ﬁlled with 20 ml of

◦ For the identiﬁcation of T. canis, sequencing of the

PBS and placed in a water bath set to 37 C. The tubes

Nemo-PCR product was performed. A single-step enzy-

were then capped to avoid evaporation, and the samples

␮

matic treatment of 5 l PCR products was carried out

were left in the water bath for 24 h after which, the 15 ml

with 2 ␮l ExoSAP-IT (USB) to inactivate excess primers and

tubes were removed, and the 50 ml tubes were capped

nucleotides.

and spun at 500 × g for 5 min. The supernatant was aspi-

Direct sequencing was performed on a 3700 auto-

rated to 10 ml, and an equal volume of 6% formalin was

mated sequencer using the Big Dye Terminator kit from

added to ﬁx any larvae present in the sample. For analysis,

Applied Biosystems, according to the manufacturer’s

samples were centrifuged at 500 × g for 5 min, the super-

instructions. Sequences were analyzed by alignment with

natant aspirated to 2 ml and transferred to 24 well plates

reference sequences using MAFFT (see http://mafft.cbrc.jp/

for larval counting using the 10× objective of an inverted alignment/server/). microscope.

Results

2.3. BAL and DNA extraction

3.1. Alignment of sequences

To obtain the BAL, lungs were washed three times with

◦ ◦

1 ml PBS (pH 7.2, 37 C), centrifuged at 400 × g, at 4 C, for Table 2 shows the alignment of sequences from the

10 min and the cell pellet was used for DNA isolation. DNA 18S rRNA gene derived from different nematodes that

from BAL was extracted using the High Pure PCR Template were retrieved from GenBank. Table 3 shows the sequence

Preparation Kit (Roche, Almere, The Netherlands) as rec- from the Nemo-PCR product from T. cati, T. canis and from

ommended by the manufacturer. the DNA isolated from BAL of infected mouse number 6

E. Pinelli et al. / Veterinary Parasitology 193 (2013) 337–341 339 a . . . . a . . . . a . . . . c . . . . t . . . .

g . . . . t . . . . g . . . . t . . . . g . . . . shown.

c . . . . a . . . . c . . . . c . . . . a . . . . are

g . . . . g . . . . a . . . . a . . . . a . . . .

c . . . . g . . . . g . . . . g . . . . c . . . .

variants c . . . . t . . . . t . . . . t . . . . t . . . .

the a . . . . g . . . . t c c g c g . . . . a . . . .

a . . . . c . . . . t . . . . g . . . . t . . . . dot,

a . . . . a . . . . t . . . . t . . . . c . . . . by

g . . . . t . . . . t . . . . t . . . . t . . . .

t . . . . c . . . . t . . . . a . . . . g . . . .

g . . . . c . . . . a . . . . a . . . . t . . . . indicated

g . . . . t . . . . g . . . . c . . a a g . . . . are

a . . . . a . c g g c . a . . t . . . . c . . . .

a . . . . t . . . . c . . . . g . . . . a . . . .

g - - - - a . . . . t . . . . c . . . . g . . . .

a . . . . g . . . . c . . . . c . . . . c . . . . nucleotides

t . . . . t . . . . g . . . . c . . . . g . . . .

a . . t t t . . . . a . . . . g . . . . g . . . .

Identical t . . . . g c c c t a . . . . g . . . . c . . . .

product.

c . . . . t . . . . a . . . . c . . . . c . . . .

c . . . . a . . . . c . . . . t . . . . a . . . .

g . . . . g . . . . c . . . . t . . . . a . . . . EF180059.

Nemo-PCR

g . . . . t . . . . a . . . . t . . . . g . . . . cati

the

t . . . . t . . . . c . . . . g . . . . t c c a a to

a . . . . c . . . . g . . . . g . . . . t . . c c Toxocara

a . . . . a . . . . t . . . . g . . . . c . . . . and

a . . . . t . . . . a . . . . c . . . . t . . . .

corresponding

c . . . . a . . . . c . . . . t . . . . g . . . . U94382

t . . . . t . . . . a . . . . a . . . . g . . . .

t . . . . a . . . . t . . . . a . . . . t . . . . canis

GenBank

g . . . . t . . . . a . . . . c . . . . a . . . .

a . . . . t . . . . a . . . . c . . . . c . . . . from

Toxocara a . . . . a . . . . t . . . . a . . . . g . . . .

t . . . . t . . . . c . . . . a . . . . c . . . .

retrieved c . . . . c . . . . g . . . . a . . . . t . . . .

U94383;

t . . . . g . . . . a . . . . a . . . . a . . . .

g . . . . a . . . . g . . . . t . . . . g . . . .

leonina t . . . . c . . . . a . . . . t . . . . t . . . .

nematodes

a . . . . a . . . . t . . . . a . . . . c . . . . various

Toxascaris

c . . . . a . . . . c . . . . g . . . . g . . . .

g . . . . c . . t t t . . . . a . . . . g a t a a from

t . . . . a . . . . t . . . . t . . . . t . . . . U94367;

gene

a . . . . t . . . . a . . . . t . . . . a . . g g

c . . . . t . . . . a . . . . a . . . . t . . . . suum

rRNA

c . . . . a . . . . t . . . . t . . . . c . . . .

18S

g . . . . c . . . . g . . . . c . t . . a . . . . Ascaris

the a . . . t . . . . g . . . . t . . . . a . . . .

sequences

a . . . c . . . . t . . . . a . . . . t . . . . from

t . . . g . . . . g . . . . c . . . . a . . . . U94365;

c . . . g . . . . t . . . g . . . . a . . . . spp. alignment

g . . c . . . . c . . . c . . . g . . . .

sequences

...... DNA g . . a . . . . . g . . . t ...... of

Anisakis

are

leonina leonina leonina leonina leonina

canis cati canis cati canis cati canis cati canis cati

spp. spp. spp. a spp. spp.

alignment suum suum suum suum suum

DNA sequences

Nematodes Anisakis Ascaris Toxascaris Toxocara Toxocara Anisakis Ascaris Toxascaris Toxocara Toxocara Anisakis Ascaris Toxascaris Toxocara Toxocara Anisakis Ascaris Toxascaris Toxocara Toxocara Anisakis Ascaris Toxascaris Toxocara Toxocara Table The The

340 E. Pinelli et al. / Veterinary Parasitology 193 (2013) 337–341

. Table 4

g . . c . .

T. canis larvae in lungs and PCR of BAL.

canis t . . g . .

Group Mouse no. Number of T. canis larvae Nemo-PCR on

g . . c . .

DNA from BAL

with Left lung Right lung

t . . g . .

−

c . . a . . 5–6 week 1 1 1

old Mice 2 3 2 +

a . . g . .

infection

− 3 0 0

a . . c . .

4 1 3 −

−

t . . a . . 500

614 +

a . . g . .

1–2 week 7 0 0 + g . . t . . experimental

old Mice 8 0 0 +

g . . c g g 930 −

t . . t . .

10 0 0 −

g . . t . . 11 0 1 + conﬁrming

12 0 0 +

c . . t . .

a . . t . . mouse,

t . . a . .

(Table 4). Molecular determination and sequence using this

c . . g . . procedure conﬁrmed that the mouse was infected with infected

T. canis. c . . c . . an

Using DNA isolated from T. canis larvae, the lower detec-

t . . c . .

tion limit using the primers and probes here described was from

g . . t . .

shown to be less than 100 fg (data not shown).

t . . c . .

a . . g . . isolated

3.2. Nemo-PCR on BAL from T. canis infected mice

g . . a . . BAL

in t . . a . .

Results from the Nemo-PCR on DNA isolated from BAL

t . . a . .

DNA

of mice infected with 1000 T. canis embryonated eggs are

of t c c c . .

shown in Table 4. T. canis larvae were recovered from the

t . . c . .

lungs of 4 of 6, 5–6-week-old mice 60 days post-infection.

a . . a . .

product Of these 6 infected mice, 2 samples of BAL were Nemo-

g . . c . .

PCR positive. DNA sequence of the Nemo-PCR product from

t . . g . . mouse number 6 conﬁrmed the presence of T. canis DNA.

t . . t . . Interestingly, although T. canis larvae were observed in Nemo-PCR

lungs of only 2 of the 6 young (1–2 weeks old) mice, the c . . a . . the

Nemo-PCR on BAL was positive for 4 of the 6 infected ani-

a . . c . .

from mals. Results from the Nemo-PCR were negative for all

t . . a . .

control uninfected mice.

a . . t . .

derived t . . a . .

4. Discussion

a . . a . .

t . . t . .

Serological antibody detection is presently the only

sequence

t . . c . .

way to conﬁrm clinical diagnosis of human toxocarosis.

the

a . . g . .

The most commonly used serological assays for detection

t . . a . .

with

of antibodies against these parasites are enzyme-linked

c . . g . .

immunoabsorbent assay and Western blot. Although these

g . . a . .

assays have been shown to have an acceptable sensitiv-

sequences a . . t . . ity and speciﬁcity (reviewed in Pinelli and Aranzamendi,

c . . c . . 2012), cross-reactions remain an important issue, par- canis

ticularly in areas endemic for different helminths. The a . . t . . T.

sequences

availability of the molecular assay described here will a . . t . . and

improve the deﬁnitive diagnosis of human toxocarosis.

t . . a . . cati

During Toxocara spp. infection larval invasion and T.

a . . a . .

alignment migration through the lungs may result in respiratory

the t . t . .

of distress characterized by wheezing, coughs and mucous

.. g t .

. .

production. Murine models for toxocarosis have been

extensively used to study the immunological and patho- canis cati canis cati

logical consequences of infection with this nematode (Buijs alignment

et al., 1994; Kayes et al., 1987; Pinelli et al., 2005, 2007). 3

DNA

Experimental infection of mice with T. canis results in pul- NematodesToxocara DNA Toxocara Mouse-BAL Toxocara Toxocara Mouse-BAL

Table

The monary inﬂammation that can persist up to 60 days post

E. Pinelli et al. / Veterinary Parasitology 193 (2013) 337–341 341

infection. Toxocara spp. larvae have been recovered from respiratory symptoms should be tested. Studies published

lungs of infected mice as early as 3 days p.i. and at 60 by others indicate that the PCR assay on BAL is highly

days p.i. few larvae could still be recovered. In BAL of accurate for diagnosing other pulmonary infections such

infected animals, inﬁltrating eosinophils as well as anti- as invasive aspergillosis (Sun et al., 2011).

bodies against Toxocara spp. excretory/secretory antigens In conclusion, we describe a novel real-time PCR that

can be detected (Pinelli et al., 2005). in combination with DNA sequencing allows detection

In the present study we used DNA isolated from BAL and identiﬁcation of T. canis and other nematodes from

ﬂuid derived from T. canis infected mice and a novel PCR the Superfamily Ascaridoidea. Preliminary ﬁndings indi-

to detect this roundworm. The Nemo-PCR here described cate that the Nemo-PCR is able to amplify T. canis DNA in

combined with DNA sequencing, has the potential to detect BAL of experimentally infected mice. The availability of a

and identify different nematodes from the Ascaridoidea validated direct molecular assay for Toxocara spp. DNA in

Superfamily including T. canis. BAL will improve diagnosis of human toxocarosis, particu-

A range of different helminths are routinely send to larly for patients with respiratory distress.

this laboratory for identiﬁcation using other primer com-

binations that ampliﬁes a region from the ITS1 to the 5.8S Conﬂict

of interest statement

rRNA gene (Gasser et al., 1993). The length of the ampliﬁed

product varies with each species but the shorter products None.

are approximately 470 bp and the larger ones well over

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