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Acta Tropica 104 (2007) 122–132

Evaluation of follow-up of therapy with fenbendazole

incorporated into stabilized liposomes and immunomodulator
glucan in mice infected with Toxocara canis larvae
G. Hrčkova a,∗ , S. Velebný a , A. Obwaller b , H. Auer b , G. Kogan c
a Parasitological Institute of the Slovak Academy of Sciences, Hlinkova 3, 04001 Košice, Slovak Republic
b Department of Medical Parasitology, Clinical Institute of Hygiene, Medical University of Vienna, Kinderspitalgasse 15, 1095 Vienna, Austria
c Institute of Chemistry, Center for Glycomics, Slovak Academy of Sciences, Dúbravská cesta 9, 845 38 Bratislava, Slovak Republic

Received 24 May 2007; received in revised form 12 July 2007; accepted 22 August 2007
Available online 26 August 2007

Abstract
Anthelmintic activity of benzimidazole carbamate anthelmintics is low against dormant Toxocara canis larvae during late infec-
tions in paratenic hosts. The present study was conducted to examine the efficacy of pure fenbendazole, or drug incorporated
into sterically stabilized liposomes (SL-FBZ) administered to T. canis-infected mice alone and after its co-administration with the
immunomodulator (1 → 3)-ß-d-glucan against larvae localized in muscles and brains. Therapy with either drug forms (in total
250 mg/kg in 10 doses) commenced on day 28 post-infection (p.i.) and the efficacy of treatment, examined on day 30 after the last
dose of drug, was the highest in groups of mice treated with SL-FBZ in combination with glucan (89.5 ± 5.8% in the muscles,
66.1 ± 8.1% in brains). During 56 days of follow-up after termination of therapy, serum levels of anti-TES IgG antibodies, circulating
IgG-TES immune complexes (CIC) as well as IgG antibodies to the most immunogenic part of recombinant myosin antigen of T.
canis larvae were investigated. In contrast to anti-TES IgG antibodies, levels of CIC and anti-myosin antibodies were in the linear
correlation with the efficacy of treatments beginning from day 38 post-therapy. We also showed that the serum levels of CIC as well
as anti-myosin IgG antibodies seem to be the suitable serological markers for the monitoring of progress in larval destruction and
TES resorption from the tissues.
© 2007 Published by Elsevier B.V.

Keywords: Toxocara canis; Fenbendazole; Glucan; Stabilized liposomes; Antibodies; Immune complexes; Myosin

1. Introduction and can survive for a long period in a developmen-

Toxocara canis can invade a wide range of hosts,
but only in the candid species larvae do mature into
the intestinal adult stage. In humans, mice and other
paratenic hosts larvae migrate via parenchymal organs
∗ Corresponding author. Tel.: +421 55 6334455;
fax: +421 55 6331414.
E-mail address: hrcka@saske.sk (G. Hrčkova).
tally arrested stage. In untreated mice of C57BL/6 and
BALB/c strains, larvae were found in brains and car-
cass, even 1 year after the inoculation with only a slight
reduction in the total larval count (Bardon et al., 1994). In
humans, infection with Toxocara spp. can be confirmed
serologically by means of detection of the specific circu-
lating antibodies and eosinophilia (Smith and Noordin,
2006; Magnaval et al., 1991). However, these tests cannot
indicate on the duration of infection, since specific IgG
antibodies are detected in the serum of patients for

0001-706X/$ – see front matter © 2007 Published by Elsevier B.V.

doi:10.1016/j.actatropica.2007.08.006
 G. Hrčkova et al. / Acta Tropica 104 (2007) 122–132
several years after initial infections even after termina-
tion of drug administration (Magnaval, 1995; Kinčeková
et al., 1999; Rubinsky-Elefant, 2004). Amongst the
drugs potentially effective for the treatment of patients,
benzimidazole carbamates (albendazole, mebendazole
and fenbendazole) and diethylcarbamazine were rec-
ommended by the WHO in 1995. Development of the
appropriate serological tests, which could monitor the
process of larval killing and their resorption from the
tissue following therapy, would allow the monitoring of
disease progress and suggest more suitable treatment.
In mice with experimental Toxocara canis infection,
Nicholas and Stewart (1979) tested the effect of several
benzimidazole carbamates, using a similar dosage as that
applied in our study. They reported that fenbendazole had
a significant larvicidal effect during the migratory phase
(days 1–9 p.i.), but was ineffective against the larvae
in brain and muscles when administered at later stages.
Similarly, Holt et al. (1981) found very limited efficacy
of fenbendazole against migrating Toxocara canis lar-
vae in the brain and carcass of infected mice. Treatment
with several benzimidazole carbamates, started 24 h p.i.,
reduced the number of larvae and caused significant
arrest of the migrating larvae in liver (Abdel-Hameed,
1984). These findings, however, are likely to have only
minimum relevance to the treatment of humans, since
patients with toxocariasis are usually diagnosed long
after most larval migration cease.
Liposomes are vesicles composed of concentri-
cally arranged phospholipid membranes, in which
both hydrophobic and hydrophilic substances can be
entrapped (Papahadjopoulos, 1993). Liposomal drug
formulations allow solubility problems to be overcomed,
act as slow-releasing drug reservoirs of the substances
with fast blood clearance and in some instances can
mediate organ or site-specific targeting of the entrapped
drug (Fielding, 1991). Significant progress in liposo-
mal technology has been made by development of
the modification of their surface phospholipids with
polyethylene-glycol (PEG), which was shown to be
very advantageous in extending the half-life of drugs
for a much longer period, whilst markedly decreasing
their clearance by cells of the mononuclear phagocytic
system (MPS) (Woodle and Lasic, 1992). Liposomes
with the entrapped drugs, so-called “stealth” liposomes,
were used for the treatment of various kinds of can-
cers (for example, Storm et al., 1998). Recently, we
have reported on significant enhancement of the ther-
apeutic effect of albendazole incorporated into stealth
liposomes in experimental Toxocara canis infection
(Hrčkova, 2006). Toxocara canis larvae can produce
high levels of excretory-secretory (TES) antigens, which
123
are either immobilised in the tissues or circulate in the
serum (Parsons et al., 1986; Maizels et al., 2006). As a
consequence, free antigens generate circulating immune
complexes (CIC) after reacting with pre-existing anti-
bodies. The benefit of detection of the circulating antigen
and immune complexes for evaluation of follow-up
of treatment was demonstrated in patients with cys-
tic echinococcosis (Craig, 1986; Bonifacino et al.,
1997). The consequence of larval degeneration fol-
lowing immune killing after the drug administration
is the release of previously covert-somatic antigens.
These could prove useful in monitoring the efficacy of
chemotherapy (Smith and Noordin, 2006). In eukaryotic
organisms, myosins are represented by a wide range of
different classes of molecules, of which class II myosins
drive muscle contractions as well as cell organization
(Nikolaou et al., 2006). In nematodes, myosin is essen-
tial for larval motility, however once liberated it becomes
a major antigen in Brugia malayi (Dissanayake et al.,
1992) or Onchocerca volvulus (Erondu and Donelsen,
1990). In the present work the recombinant protein frag-
ment corresponding to the most immunoreactive region
of myosin (Obwaller et al., 2001) was used in the sero-
logical assay.
Co-administration of the immunomodulator polysac-
charide derived from the yeast cell walls – glucan – with
anthelmintics was shown to be beneficial for the activa-
tion of the host immune response during nematode and
cestode infections, which contributed to a significantly
enhanced efficacy of the drugs (Velebný et al., 1997;
Hrčkova et al., 2007). In the present study, the effect
of combined therapy with fenbendazole in stealth lipo-
somes and glucan was evaluated in experimental murine
toxocariasis during the later stage of infection. The effi-
cacy of the drug formulations was determined based on
the reduction of larval counts in the brains and mus-
cles of mice. In addition, follow-up of the therapy was
assessed by means of monitoring the levels of circulat-
ing IgG antibodies to TES antigens, levels of immune
complexes as well as on the basis of circulating IgG
reacting with Toxocara canis recombinant myosin frag-
ment.
2. Materials and methods
2.1. Drugs and their formulations
Fenbendazole (FBZ) (Sigma Chemical Co., St.
Louis, MO, USA) was suspended in saline containing
0.4% Tween 80 (SIGMA) giving rise to a suspen-
sion with a concentration of 2.5 mg/mL. Stabilised
liposomes with entrapped FBZ (SL-FBZ) were pre-
124 G. Hrčkova et al. / Acta Tropica 104 (2007) 122–132
pared as previously described (Velebný et al., 2000b).
Concentration of FBZ (2.40 mg/ml of liposomal suspen-
sion) was determined spectrophotometrically at 353 nm,
representing 85% of the entrapment of the initial
amount. Carboxymethylated (1 → 3)-ß-d-glucan (CM-
glucan) from Saccharomyces cerevisiae was prepared
according to a procedure described by Machová et al.
(1995).
2.2. Infection of animals and experimental design
Inbred male mice of C57BL6 strain were divided
in four groups each comprising 15 animals. Toxocara
canis eggs were obtained by dissection of gravid females,
taken from the naturally infected puppies. After purifica-
tion, the eggs were embryonated in 0.1N H2 SO4 at room
temperature and preserved at 4 ◦ C until used. Mice were
inoculated with 1000 Toxocara canis eggs per animal by
a gastric tube and untreated mice served as the control
(group 1). Administration of the drugs started on day 28
post-infection (p.i.). FBZ in suspension (group 2) and
FBZ in stabilized liposomes (SL-FBZ) (group 3) were
administered orally or subcutaneously, respectively at a
dose of 25 mg/kg body weight twice a day for 5 con-
secutive days (in total 250 mg/kg in 10 doses). Mice in
group 4 were treated with the same doses of SL-FBZ
as in group 3, and they received in addition two doses
of glucan (one dose comprising 5 mg/kg body weight)
intramuscularly on days 25 and 28 p.i.
2.3. Recovery of Toxocara canis larvae and
evaluation of drug efficacy
Ether-anaesthetised animals were sacrificed by cer-
vical dislocation on day 30 after the last dose of drug.
Toxocara larvae from the brain were isolated by Baer-
man’s method (based on the phenomenon of thermo-
and hydrotropisms). The musculature of the whole body
has been digested in artificial digesting juice, containing
1% pepsin (10,000 U) and 0.45% hydrochloric acid to
liberate muscle-dwelling larvae. The efficacy of prepa-
rations (in %) in the brain and in musculature of mice
was calculated from the numbers of isolated larvae from
treated and control groups of mice (n = 8 for each group)
according to the formula:
100 × (NC − NT )
Efficacy(%) =
NC
NC , larval counts in control group/gram of tissue; NT ,
larval counts in treated groups/gram of tissue.
2.4. Preparation of TES antigens
Toxocara canis larvae were cultivated according
to the modified method of De Savigny (1975) in
RMPI-1640 medium (Sigma), supplemented with 1%
glutamine, antibiotics and amphothericine at 37 ◦ C in an
atmosphere containing 5% CO2 . The supernatants were
harvested weekly, filtered, pooled and dialysed against
0.1 mM PBS on a 5 kDa cut-off dialysis tube (Sigma).
Afterwards, liquid antigen was lyophilised and stored at
−70 ◦ C until used. Protein concentration in mg of TES
antigen powder was determined by the Bradford protein
assay (Bio-Rad, Hertfordshire, UK) using BSA as a pro-
tein standard (Sigma) according to the manufacturer’s
instructions.
2.5. Production and isolation of IgG fraction from
hyperimmune sera to TES
Two New Zealand rabbits of about 3 kg body weight
were used in the immunisation protocol (Johnstone and
Thorpe, 1996) with purified larval TES antigens and
collection of blood was performed weekly. IgG frac-
tion from serum was isolated from supernatant following
precipitation with ammonium sulphate, which was then
subjected to a final separation by affinity chromatogra-
phy using Affi-Prep protein A (Bio-Rad). After protein
determination, IgG fractions specific to TES antigens
were aliquoted and kept at −70 ◦ C until used.
2.6. Preparation of recombinant myosin fragment
Cloning of cDNA of Toxocara canis myosin B heavy
chain, expression of individual recombinant fragments
and analysis of the immune response against each of
them was described in details previously (Obwaller et
al., 2001). The authors found that the main immunoreac-
tive region was the carboxy-terminal half of the myosin
head region (fragment Tcmyofr2), hence a correspond-
ing recombinant fusion protein was used in the present
study. Briefly, DNA sequence coding this fragment was
ligated into the vector pPHCy1A cleaved with Age1 and
Kpnl and subsequently transformed into Escherichia coli
XL1-Blue resulting in the expression plasmid (Obwaller
et al., 2001). This plasmid was then transformed into
E. coli BL21 (DE3) plysS (Stratagene, La Jolla, USA).
The cell supernatants, after their lysis, were collected
and the fusion protein was purified by Ni-chelate affin-
ity chromatography (Qiagene, Valencia, CA, USA). The
molecular weight (MW) of this fusion protein estimated
by SDS-PAGE electrophoresis was 37 kDa, of which the
MW of myosin fragment was 13.5 kDa. The cytohesin-1
 G. Hrčkova et al. / Acta Tropica 104 (2007) 122–132
PH domain from pPHCy1A vector without insert was
expressed and purified in the same way.
2.7. ELISA test for determination of IgG antibodies
in the serum
Enzyme-linked immunosorbent assay (ELISA) for
detection of IgG serum antibodies to TES antigen and
to recombinant myosin fragment was performed in 96-
well maxi-sorb plates (Nalgene Nunc, Hereford, UK).
Plates were sensitised with either TES antigen at a pro-
tein concentration of 3 or 10 ␮g/mL of recombinant
myosin fragment, both in 0.05 M sodium bicarbonate
buffer (pH 9.6; 100 ␮L/well). Coating was performed
for 2 h at 37 ◦ C following overnight incubation at 4 ◦ C.
Pooled serum samples obtained from 6-15 mice were
used at a dilution of 1:100 and secondary antibodies (goat
anti-mouse IgG-Px, Sigma) at a dilution of 1:7000, both
diluted in PBS supplemented with 2% BSA and 0.05%
Tween-20 (pH 7.2). Incubations with sera and conju-
gate were performed at 37 ◦ C for 1 h and, after washing,
the diluted substrate (o-phenylene-diamine, Sigma) and
hydrogen peroxide was added to each well. The reac-
tion was stopped with 2N H2 SO4 and optical density
values were measured at 492 nm (Multiscan Plus, Ani
Labsystem Oy, Vantaa, Finland).
2.8. Sodium dodecyl sulphate polyacrylamide gel
electrophoresis (SDS-PAGE) and Western blot
analysis
Recombinant antigen was dissolved in 0.5 M
Tris–HCl puffer (pH 6.8, Bio-Rad Instruction Manual) at
a concentration of 8 ␮g/10 ␮L, subjected to SDS-PAGE
on a 12% gel and then electro-transferred to nitrocelul-
lose membrane (Millipore, Billerica, MA, USA).
The immunoblotting procedure for detection of
IgG antibodies to recombinant myosin fragment was
described previously (Obwaller et al., 2001). Mice sera
were diluted 1:500 and incubated overnight at 4 ◦ C. After
washing with PBS without BSA, blots were exposed
to alkaline-phosphatase coupled anti-mouse IgG conju-
gate (Jackson ImmuneResearch, West Grove, PA, USA)
for 1 h at 37 ◦ C. The enzyme reaction was visualised
by using nitro blue tetrazolium and 5-bromo-4-chloro-
indolyl phosphate (Sigma) as substrate.
2.9. ELISA for detection of circulating immune
complexes (CIC)
The levels of CIC in the sera of mice were moni-
tored by a modified method described by Arias-Bouda
125
et al. (2003). Maxi-sorb microtitre plates were coated
with rabbit anti-TES IgG antibodies at a concentration of
9 ␮g/mL in 0.15 M PBS, pH 8.0 (100 ␮L/well) and incu-
bated overnight at 37 ◦ C. After blocking the plate for 1 h
with 1% BSA in PBS (pH 8.0), 100 ␮L of serum samples
diluted 1:100 in dilution buffer (0.1 M Tris, 0.15 NaCl,
1% BSA, 0.05% Tween 20, pH 8.0) was added to each
well in quadruplicates. The plate was incubated for 3 h
at 37 ◦ C with gentle shaking followed by 5-fold washing
and then secondary antibody, peroxidase-labeled goat-
anti-mouse IgG (affinity purified, Sigma) diluted 1:5000
in dilution buffer, was added. Binding of conjugate to the
antibody portion of CIC was allowed for 2 h at 37 ◦ C and
the colour reaction was developed as described above.
The specificity of CIC binding to rabbit anti-TES IgG
was tested with sera from healthy mice and mean OD
value was subtracted from OD values for infected mice.
In another control reaction for non-specific binding of
the conjugate to rabbit anti-TES IgG antibodies, mouse
serum as a source of CIC, was omitted in the assay. More-
over, non-specific binding of goat-anti mouse IgG-Px to
TES (if present in CIC) was examined in an ELISA reac-
tion in which wells were sensitised with TES in serial
dilutions (0.5–3 ␮g/mL) in sodium bicarbonate buffer as
described previously, followed by incubation with con-
jugate. The mean OD values (n = 4) were subtracted from
OD values for each serum sample.
2.10. Statistical evaluation
For the statistical evaluation of differences between
larval numbers in control and treated groups of
mice and also between all treated groups separately,
Kruskal–Wallis ANOVA test and Mann–Whitney U-test
were used. P level of significance in indicated in the
tables. Homogenity of serological data distribution on
each time point was analysed by F-test for small sam-
ples. Statistical differences within all four groups were
assessed using one-way ANOVA, followed by post hoc
Tukey’s test (P level indicated in text), which allowed
comparison between each two groups at each time-point.
Analysis was performed using the Statistica 6.0 (Stat
Soft, Tulsa, USA) statistical package.
3. Results
3.1. Effect of the treatment on reduction of
Toxocara canis larvae in the muscles and brains
The larval counts and the efficacy of treatment with
FBZ in suspension as well as FBZ in stabilised lipo-
somes (SL-FBZ) alone and in combination with glucan
126 G. Hrčkova et al. / Acta Tropica 104 (2007) 122–132

Table 1
Recovery of Toxocara canis larvae in the brains and muscles of mice and efficacy of treatment (in %) with fenbendazole (FBZ) in suspension as
well as FBZ in stabilised liposomes (SL-FBZ) alone and in combination with glucan
Groups of mice/drug formulation Brains Muscles
administered
Number of larvae ± S.E.M. Efficacy of treatment Number of larvae ± S.E.M. Efficacy of treatment

Control 142.2 ± 18.7 – 224.8 ± 29.0 –

FBZ 114.8 ± 16.1§ 19.3% 152.2 ± 25.8* 32.3%
SL-FBZ 69.4 ± 12.7* 59.2% 68.3 ± 15.8* 69.6%
SL-FBZ + glucan 48.2 ± 11.2* 66.1% 23.6 ± 9.3* 89.5%

n = 8 (for each group). Statistical evaluation of the significance was performed by Kruskal–Wallis test and Mann–Whitney U-test.
* Significantly different number of larvae between control and treated group of mice at P level < 0.001.
§ Significantly different number of larvae between control and treated group of mice at P level < 0.05.

Table 2
Comparison of the statistical difference between numbers of recovered larvae from the brains of treated groups of mice necropsied at day 30 after
administration of the last dose of drug
Number of larvae in the FBZ compared FBZ compared with SL-FBZ SL-FBZ compared with SL-FBZ
brains of mice with SL-FBZ plus glucan plus glucan

114.8 ± 16.1 69.4 ± 12.7 114.8 ± 16.1 48.2 ± 11.2 69.4 ± 12.7 48.2 ± 11.2
P level of significance P < 0.001 P < 0.001 P < 0.01

Table 3
Comparison of the statistical difference between numbers of recovered larvae from the muscles of treated groups of mice necropsied at day 30 after
administration of the last dose of drug
Number of larvae in the FBZ compared FBZ compared with SL-FBZ SL-FBZ compared with SL-FBZ
muscles of mice with SL-FBZ plus glucan plus glucan

152.2 ± 25.8 68.3 ± 15.8 152.2 ± 16.1 23.6 ± 9.3 68.3 ± 15.8 23.6 ± 9.3
P level of significance P < 0.001 P < 0.001 P < 0.001

was evaluated in the muscles and brains of mice on day

30 post-therapy (p.t.) (day 62 p.i) (Table 1). The reduc-
tion of larvae numbers and the efficacy of the treatment
(in %) was higher against muscle larvae than on lar-
vae found in the brains. The highest reduction of larval
counts was observed in the group of mice, which were
treated with SL-FBZ in combination with glucan (89.5%
in the muscles, 66.1% in brains). In addition, significant
differences between larval numbers in brains (Table 2)
and muscles (Table 3) were found after comparison of
each two pair of treated groups of mice.

3.2. IgG antibody response to TES antigens after

the therapy

The levels of anti-TES IgG antibodies in the serum of
mice from control and treated groups during the follow-
up of the therapy are shown in Fig. 1. The mean cut-off
value of antibody titres in ELISA obtained with normal
mice serum was 0.068 ± 0.0047. Whereas in untreated
mice (group 1), antibody levels gradually increased with
Fig. 1. Levels of IgG antibodies specific to T. canis TES antigens in
infected untreated groups of mice and in mice within the follow-up
of therapy with FBZ alone, FBZ in stabilized liposomes (SL-FBZ)
and with this drug form in combination with glucan. The data rep-
resent the mean absorbance ± S.E.M. obtained in ELISA and pooled
serum samples were tested in quadruplicates. ELISA was performed
two times.
 G. Hrčkova et al. / Acta Tropica 104 (2007) 122–132 127

time with the highest recorded OD value reached on day

88 p.i. (1.569 ± 0.131), administration of individual drug
formulations markedly modulated the dynamics of IgG
antibodies specific to TES antigens. Significantly lower
(P < 0.01) antibody levels during the whole follow-up
in comparison with control were observed in the group
treated with SL-FBZ alone. Administration of pure FBZ
resulted in the lowering of anti-TES IgG titres lately,
from day 10 p.t. (0.831 ± 0.064; P < 0.05, time-paired
with control) and were significantly lower than in control
group during the follow up (P < 0.001). After adminis-
tration of SL-FBZ in combination with glucan, specific
antibodies declined rapidly on day 1 p.t. (0.623 ± 0.075,
Fig. 2. Changes in the levels of circulating T. canis TES-IgG immune
P < 0.01, time-paired with control) followed by the
complexes (CIC) in the serum of infected untreated group of mice
elevation, peaking on day 30 p.t. (1.169 ± 0.121). In and in mice within the follow-up of therapy with FBZ alone, FBZ
contrast to the other treated groups, a gradual decline in stabilized liposomes (SL-FBZ) and with this drug form in com-
of anti-TES IgG antibodies was recorded in this group bination with glucan. Data for each time point are expressed as mean
beginning from this day (day 38 p.t. 1.158 ± 0.106; OD ± S.E.M. Determination of CIC was performed by single immune-
complex ELISA assay in 96-wells plates coated with rabbit anti-TES
day 56 p.t. 1.126 ± 0.098, P < 0.001, time-paired with
IgG in concentration of 9 ␮g/mL.
control).

3.3. Levels of specific circulating TES-IgG immune (fragment Tcmyofr2, Obwaller et al., 2001) were eval-
complexes uated in a sandwich ELISA assay. Mean absorbance
values found for sera during the follow-up of the ther-
A single-immune complex ELISA test was used apy and in the control group are presented in Fig. 3. In
in this study to examine whether levels of circulating the control group, the similar low levels of specific anti-
immune complexes (CIC) can reflect inhibition of TES myosin antibodies were found within 88 days p.i. (day 1
synthesis by larvae and the process of larval destruc- p.t., 0.412 ± 0.035; day 56 p.t., 0.320 ± 0.040). Follow-
tion following the therapy. The mean cut-off level was ing FBZ and SL-FBZ drug administration, concentration
0.105 ± 0.045, the value obtained after subtracting mean of circulating antibodies to recombinant myosin frag-
OD values for negative controls (see Section 2). In ment temporarily increased peaking on day 38 p.t.
the control group, CIC levels raised rapidly within
14 days p.i. (1.148 ± 0.122), then persisted in similar
concentrations showing a small fluctuation within the
entire experiment (Fig. 2). On the other hand, admin-
istration of either drug formulation markedly affected
concentration of CIC that peaked on day 23 p.t. (55
p.i.) after therapy with FBZ (1.392 ± 0.111, P < 0.001)
and SL-FBZ (1.475 ± 0.123, P < 0.001, time-paired with
control). Co-administration of glucan with SL-FBZ to
mice in the group 4 resulted in the temporal and insignif-
icant elevation over that in control between day 1 p.t.
(1.495 ± 0.099) and 10 p.t. (1.249 ± 0.048). From this
day, CIC levels declined rapidly up to day 38 p.t. (70
p.i.), when very low titre was detected (0.344 ± 0.034,
P < 0.001).
Fig. 3. Serum levels of IgG antibodies reacting with recombinant pro-
3.4. Effect of chemotherapy on Toxocara canis tein fragment of T. canis myosin (fragment Tcmyofr2, Obwaller et al.,
2001) evaluated in a sandwich ELISA assay. Data for each time point
anti-myosin IgG antibodies
represent mean absorbance ± S.E.M. for serum samples obtained from
infected untreated groups of mice and from mice within the follow-up
Levels of circulating IgG antibodies to the most of therapy with FBZ alone, FBZ in stabilized liposomes (SL-FBZ) and
immunogenic part of T. canis somatic protein myosin with this drug form in combination with glucan.
128 G. Hrčkova et al. / Acta Tropica 104 (2007) 122–132

Fig. 4. (A–D) Western blot reactivity of recombinant myosin antigen (fragment Tcmyofr2 used in concentration 8 ␮g/␮L) of T. canis probed with
sera obtained on days 5, 10, 23, 30, 38 and 56 post-therapy. A, control untreated mice; B, mice treated with FBZ alone; C, mice treated with FBZ
in stabilized liposomes (SL-FBZ); D, groups of mice treated with SL-FBZ in combination with glucan. MW of fusion recombinant antigen was
37 kDa.

(70 p.i.) (FBZ, 0.567 ± 0.055; SL-FBZ, 0.806 ± 0.084,
P < 0.001, time-paired with control) showing some
fluctuations. The most intense antibody response was
elicited after treatment with SL-FBZ in combina-
tion with glucan, being the highest on day 38 p.t.
(1.435 ± 0.142, P < 0.001), then a rapid decline was
recorded (day 56 p.t., 0.147 ± 0.02, P < 0.001, time-
paired with control).
Specificity and amount of IgG antibodies to myosin
fusion protein fragment was tested by the Western blot
technique using sera from individual groups of mice on
selected days p.t. (Fig. 4A–D). The intensity of this band
reflected the concentration of circulating antibodies to
myosin fragment and was in good correlation with the
titres determined in ELISA test.
4. Discussion
Human toxocariasis is a chronic infection, which may
last for number of years and the reactivated migration
of dormant tissue larvae can occur at any time. Effi-
cacy of the treatment in patients can be assessed only
indirectly from the decline of clinical symptoms and
eosinophilia as the levels of specific anti-TES antibodies
usually persist for a long period, even in patients with
much improved clinical parameters (Magnaval, 1995;
Łuźna-Lyskov et al., 2000).
In the present study, we showed that treatment
with fenbendazole, incorporated into liposomal carriers
which contain PEG-derivatised phospholipids (Allen et
al., 1989), was much more effective on T. canis larvae
in the brains (59.2 ± 6.2%) and muscles (69.6 ± 5.0%)
during the late stage of experimental T. canis infec-
tion than administration of pure FBZ (19.3 ± 9.5% in
brains, 32.3 ± 4.0% in muscles). These kinds of carriers,
referred to as sterically stabilized or “stealth” liposomes
(SL) were shown to maintain therapeutical plasma levels
of incorporated drug for more than 1 week after a sin-
gle administration in comparison with the conventional
liposomes and free drug (Woodle and Lasic, 1992). Ben-
zimidazole carbamates are relatively insoluble in water,
which leads to poor oral absorption ranging from 1–5%
in humans to 20–30% in mice peaking after 2–3 h p.i.
(Gottschall et al., 1990). Early treatment with FBZ or
ABZ (100 mg/kg body weight) between days 2–7 p.i.
resulted in the significant retention of most of larvae in
the liver, however the same dose of drugs given between
days 8–13 p.i., when larval migration is nearly com-
pleted, had no effect (Abo-Shehada and Herbert, 1984).
We assume that the low anthelmintic effect of benzimi-
dazole carbamates on these dormant larvae is probably a
result of low sub-curative plasma concentrations of drug.
Although the physiology of hypobiotic dormant tissue
larvae is not well understood, the possibility also exists
 G. Hrčkova et al. / Acta Tropica 104 (2007) 122–132
that the dormant larvae are less sensitive to anthelmintic
activity of these drugs than the migrating larvae.
In our previous studies on experimental murine tox-
ocariasis (Velebný et al., 2000a; Hrčkova and Velebný,
2001), the similar dosage of FBZ as used in the present
work was administered subcutaneously entrapped in the
conventional liposomes and showed efficacy up to 55%
against muscle larvae and up to 44% on larvae in the
brains. In this respect, we suppose that administration of
FBZ in sterically stabilized liposomes, which are more
resistant to the degradation by the cells of MPS than
the conventional liposomes (Storm et al., 1998), resulted
in the prolonged circulation of the therapeutic plasma
levels of FBZ in the serum and tissues due to the grad-
ual release of drug from liposomes. In accordance with
our hypothesis, New et al. (1994) and Dvorožňáková
et al. (2004) reported an increased biological availabil-
ity and efficacy of albendazole entrapped in liposome
carriers compared with the free drug administered to
mice with experimental echinococcosis. In the group
treated with SL-FBZ in combination with two doses
of immunomodulator glucan, the efficacy on larvae in
the muscles and brains was much higher (89.5 ± 5.8
and 66.1 ± 8.1%, respectively) than in other treated
groups. ␤-d-Glucans are polysaccharides of natural ori-
gin with an ability to act as non-specific modulators of the
immune system (Williams, 1997; Kogan, 2000). There
are numerous studies upon their stimulatory activity on
various immunity mechanisms during the carcinogene-
sis, viral, bacterial and infectious diseases. Recently, we
(Hrčkova et al., 2007) have shown that co-administration
of anthelmintic drug praziquantel with liposomized ␤-
d-glucan can enhance the damaging action of the drug
on the larvae of Mecocestoides vogae as the result of
activation of macrophage effector functions and produc-
tion of more cytotoxic antibodies. Šoltýs et al. (1996)
reported that in mice with experimental T. canis infection
administration of ␤-d-glucan with IgG and zinc caused
significant reduction of larval numbers and moderately
elevated IgG levels. A similar synergistic effect of a com-
bination of SL-FBZ and ␤-d-glucan demonstrated in the
present study seems to be in a good agreement with the
previous findings.
Toxocara canis larvae possess very high metabolic
capacity in vitro and in vivo, producing large amounts
of excretory and secretory molecules (TES antigens)
(De Savigny, 1975; Maizels et al., 1984), which induce
a strong humoral immune response. Indeed, levels of
anti-TES IgG antibodies were increasing within nearly
3 months from the beginning of our experiment. FBZ
treatment significantly (P < 0.01) reduced titres of cir-
culating antibodies, whilst even stronger reduction was
129
observed when FBZ was administered in stealth lipo-
somes. Smaller decline of IgG antibodies was recorded
after administration of pure mebendazole (Bardon et
al., 1995) and albendazole in conventional liposomes
(Dvorožňáková et al., 1998) during T. canis infection
in the laboratory mice. Also, in patients suspected to
be suffering from toxocariasis, anti-TES antibody titres
declined very slowly, and some authors (Luo et al., 1999)
suggested that these results could not unambiguously
certify the parasites were still alive. It is well known
that anthelmintic activity of benzimidazole carbamates
is based on paralysis of worm cells’ homeostasis as
the result of specific disruption of microtubules (Lacey,
1988). Therefore, the direct activating effect of these
drugs on the immune response does not seem to be
plausible. The observed decrease of anti-TES antibod-
ies titre in the treated groups might be a consequence
of inhibition of larval metabolic activity due to larval
paralysis/death, which was more extensive after SL-
FBZ administration than after application of FBZ alone.
During the migration of larvae, large amounts of TES
antigens are deposited in the liver, lungs, muscles and
brains, where they can persist for long periods of time
in the infected animals (Parsons et al., 1986) and in
patients diagnosed from biopsies post-mortem (Hill et
al., 1985). In our study, temporary though significantly
elevated levels of specific antibodies were observed in
the group treated with SL-FBZ in combination with glu-
can in comparison with the control group. We assume
that glucan co-administration triggered the cascade of
immune events, result of which was elimination of TES
deposits from the tissues by antigen-presenting cells and
consequently the temporal elevation of anti-TES IgG
antibody titres.
Immune complexes and free antigens circulate in
serum during infection diseases and can provide addi-
tional and more accurate information on the follow-up
of the disease and/or treatment (Bonifacino et al., 1997;
Ferragut et al., 1998). In the present study, we monitored
IgG-TES immuno-complexes, levels of which increased
rapidly within 14 days p.i. and persisted in the similar
concentrations in the sera of control mice. In contrast,
dynamics of CIC levels has been markedly modulated
in mice of all treated groups, however the levels of CIC
showed very similar time-profile dependence as did anti-
TES IgG antibodies in the FBZ and SL-FBZ treated
groups (Fig. 2). Interestingly, the rapid elevation of CIC
levels following the last dose application was observed
in the group receiving SL-FBZ and glucan, the treatment
showing the strongest larvicidal effect. These CICs were
probably formed by TES released from dead larvae and
the tissues with pre-existing IgG antibodies. Glucan can
130 G. Hrčkova et al. / Acta Tropica 104 (2007) 122–132
bind to the several receptors on monocytes/macrophages,
which in turns stimulates their non-specific effector func-
tions such as phagocytosis and production of reactive
oxygen species (Williams et al., 1996). Almost complete
disappearance of CIC was observed at the end of the
experiment in this group indicating that efficient clear-
ance of CIC by activated immune cells took place. In
other groups of mice not subjected to glucan treatment,
larval destruction and liberation of TES seems to take
much longer time basing on the appearance of marked
CIC levels on day 55 p.i. (23 p.t.). In our and other
experimental studies, anti-TES IgG antibodies were pre-
sented in higher concentrations than CIC in the untreated
animals, and a similar linear correlation was observed
between the decrease of T. canis larval counts and the
levels of CIC following the therapy with benzimidazole
carbamates (Bardon et al., 1995; Cuellar et al., 1990;
Hrčkova, 2006).
Antibodies to the abundant somatic nematode anti-
gen myosin, represent an additional serological marker,
which could be used as a tool for monitoring the follow-
up of treatment. In human donors infected with nematode
Brugia malayi, the overall sero-positivity to the two
clones of recombinant myosin was approximately 32%
and native antigen was identified in somatic extracts
of larvae and adults of this nematode (Dissanayake et
al., 1992). In the present study we assayed a recom-
binant T. canis myosin fragment, which showed the
highest positive response with 73% of human toxocari-
asis sera (Obwaller et al., 2001). In the untreated group
very low IgG immunoreactivity was detected which was
probably elicited after myosin leakage from the larvae
damaged by the host immune system as this antigen is not
actively secreted (unpublished observations). Levels of
anti-myosin antibodies increased in time-period between
days 55–70 p.i. (23–38 p.t.) and the titres were in a linear
correlation with the efficacy of drug formulations. These
results are consistent with the findings of the previous
serological tests, whilst the most pronounced temporary
IgG elevation was recorded after a combined treatment
with SL-FBZ and glucan. This might indicate that mas-
sive resorption of killed larvae by macrophages takes
place, followed by the processing of myosin antigen to
the lymphocytes. We hypothesise that the decline of anti-
myosin antibody levels at the end of experiment indicates
that elimination of larval debris was nearly completed.
The studies presented here indicate that the efficacy
of anthelmintic fenbendazole towards to the dormant T.
canis larvae localised in the muscles and brains of exper-
imentally infected mice can be markedly augmented
when drug is administered to animals in liposomal carri-
ers in combination with the immunomodulator glucan. In
this respect, the most suitable are liposomes, which con-
tain PEG-derivatised phospholipids, as the consequence
of therapeutic plasma levels of the drug being presented
for extended period in serum. In addition, we have shown
that the serum levels of CIC as well as anti-myosin IgG
antibodies could provide additional information about
the process of larval destruction and TES resorption from
tissues. In contrast to anti-TES IgG antibodies, levels of
CIC and anti-myosin antibodies were in the linear cor-
relation with the efficacy of treatments beginning from
day 38 post-therapy.
Acknowledgements
This work was supported by SAIA—Action Austria-
Slovakia (grant no. 41s5) and partially by the Scientific
Grant Agency VEGA of the Ministry of Education of
Slovak Republic and The Slovak Academy of Sciences
(grant no. 2/7188/27 and 2/7033/7).
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