Cross-reactivity between antigens of Anisakis simplex s.l. and other

Article available at http://www.parasite-journal.org or http://dx.doi.org/10.1051/parasite/2004112219
CROSS-REACTIVITY BETWEEN ANTIGENS OF ANISAKIS SIMPLEX S.L.
AND OTHER ASCARID NEMATODES
LOZANO MALDONADO J.*, MARTIN HITA L.*, DIAZ SÂEZ V.*, MANAS ALMENDROS I.*,
VALERO LOPEZ A.* & CAMPOS BUENO M.*
Summary:
R é s u m é : RÉACTION CROISÉE D E S ANTIGÈNES D'ANISAKIS
A study of the cross-reactivity among somatic and excretory-
D'AUTRES NÉMATODES ASCARIDÉS
secretory antigens of the third stage larvae of Anisakis
simplex s.l.
SIMPLEX S.L. E T
Dans ce travail nous étudions les réactions croisées des antigènes
and somatic antigens of other ascarid nematodes (Ascaris
du troisième stade larvaire d'Anisakis simplex s.l. et des antigènes
lumbricoides,
somatiques d'autres nématodes ascarides par la technique
A. suum, Toxocara canis, Anisakis
Hysterothylacium
physeteris,
aduncum and H. fabri) was carried out by
immunoblotting. It was revealed a high degree of cross-reactivity
among ascarids in the 3 0 and > 2 1 2 kDa range by using sera
against somatic and excretory-secretory antigens of A. simplex s.l.
It has been revealed also specific components of the Anisakis
genus (< 7.2, 9, 19 and 25 kDa) that will be interesting in
diagnosis.
d'immunoblot (Ascaris lumbricoides, A. suum, Toxocara canis,
Anisakis physeteris, Hysterothylacium aduncum et H. fabri). On a
ainsi révélé une très importante réactivité croisée parmi des
ascarides
212
(antigènes somatiques et antigènes excrétés-sécrétés ou
métaboliques).
spécifiques
KEY W O R D S : ascarids, antigen, immunoblotting, cross-reacton
dans les bandes de 30 et dans celles supérieures à
kDa en employant des immunsérums anti-A. simplex s.l.
De même, nous avons révélé des fractions
(< 7.2,
9,
19 et 25 kDa) dans le genre Anisakis qui
seront intéressantes dans le diagnostic.
MOTS CLÉS : ascaridés, antigène, immunoblotting, réaction croisée.
O
n e o f the main d r a w b a c k s o f immunological
diagnosis o f parasite infection is caused b y the
complexity o f the parasite antigens, especially
the existence o f antigens shared b e t w e e n species with
varying degrees o f phylogenetic relationship and betw e e n the parasite a n d host (Capron et al, 1 9 6 8 ; Pascual et al., 1997; Favre et al., 1 9 9 8 ) . Immunological
cross-reactions a m o n g ascarid n e m a t o d e s are widespread and infection b y a species could potentially
affect on the nature o f the immune response by a n o ther species and produce false positives in the diagnosis (Kennedy et al., 1988; Kennedy et al, 1989; Iglesias et al., 1996; Nunes et al., 1997; Fernandez et al.,
1998; Lorenzo et al, 1999; Kennedy, 2 0 0 0 ) . Anisakiasis o f the human alimentary tract is frequently caused by the ingestion o f raw or u n d e r c o o k e d fish
infected b y Anisakis simplex larvae (L3) (Van Thiel et
al, 1 9 6 0 ; Ishikura et al., 1 9 9 2 ) . O n the other hand,
recently several human infections d u e to A.
simplex
larvae have b e e n reported in Spain (Domínguez Ortega
et al., 2 0 0 0 ; D a s c h n e r et al., 2000; Castan et al., 2 0 0 2 ) .
For this reason, in the present work, w e have studied
the cross-reactivity b e t w e e n somatic (SA) or excretorysecretory (ES) antigens o f the third stage larvae o f A. simplex s.l., a n d other ascarid nematodes ( A s c a r i s lumbricoides, A. suum, Toxocara
canis, Anisakis
physeteris,
Hysterothylacium
aduncum
and H. fabri) using immunoblotting to detect specific antigens that could b e
applied to serologic diagnosis in human anisakiasis.
MATERIALS AND METHODS
BIOLOGICAL MATERIAL
L
3 larvae o f the following nematodes w e r e isolated: A. simplex sensu lato (s.l.), (blue whiting,
Micromesistius
poutassou),
H. aduncum
(horse
mackerel, Trachurus
trachurus),
A. physeteris
(hake,
Merluccius
merluccius),
H. fabri (striped mullet, Mullus
barbatus
and striped red mullet, M. surmuletus).
The
hosts are given in parentheses.
Adults o f T. canis, A. suum and A. lumbricoides
were
obtained from dogs, pigs a n d humans, respectively.
* Departamento de Parasitología, Facultad de Farmacia, Universidad
de Granada. Campus Universitario Cartuja. 18071 Granada, Spain.
Correspondence: Dr. Josefa Lozano Maldonado.
Tel.: +34 958 24 38 61- Fax: +34 958 24 38 62.
E-mail: jlozano@ugr.es
ANTIGENS
Somatic antigens (SA) o f the larvae and adults helminths: all the helminths w e r e w a s h e d several times
219
with phosphate saline buffer ( P B S ) pH 7.2, and h o m o genized in PBS supplemented with protease inhibitors:
5 mM ethylenediaminetetraacetic acid, 1 mM phenylmethylsulphonyl fluoride and 0.1 trypsin inhibitor unit/
ml aprotinin (Sigma). T h e h o m o g e n a t e s w e r e applied
to several cycles o f ultrasound sonicator and centrifuged! at 2 0 , 0 0 0 r/min for 30 min at 4 ° C . T h e supernatant was harvested and conserved at - 70° C until
use.
Excretory-secretory antigens o f A. simplex
s.l. (ES):
viable L3 larvae o f A. simplex
s.l. w e r e p l a c e d in an
antibiotic-antifungal solution and axenized (Iglesias et
al., 1 9 9 7 ) . T h e n , 2 0 0 - 2 5 0 larvae/2,5 ml w e r e placed in
culture flask containing sterile saline solution at 0.9 %,
and maintained at 37° C and 5 % C O , (Valero et al.,
2 0 0 3 ) . T h e w o r m s were o b s e r v e d daily for mobility,
moulting and survival; after 7-9 days of culture, the
larvae w e r e separated and the supernatant was filtered
and centrifuged at 1,900 g for 30 min at 4 ° C and stored
at - 70° C until use.
T h e protein concentration o f the antigens was determined by the m e t h o d of Lowry (Lowry et al.. 1 9 5 1 ) .
A N T I - S A AND ANTI-ES IMMUNE SERA
T o obtain anti-SA and anti-ES sera, N e w Zeeland rabbits weighing about 2 kg were immunized intramuscularly with doses o f 1 ml/rabbit o f a 1:1 (v/v) mixture of c o m p l e t e F r e u n d ' s adjuvant and a solution
containing 6 mg of antigen. Both at 21 and 31 days
the s a m e antigen d o s e e x c e p t adjuvant was injected.
After o n e month from the last immunization, they
w e r e administered a b o o s t e r dose and the animals
w e r e bled at seven days. For each antiserum, a pool
with immune sera (from three rabbits) obtained from
successive bleedings was d o n e . Before the first inoculation, the rabbits w e r e bled via incision into the
marginal vein of the ear, using this serum as a negative control.
IMMUNOBLOTTING
T h e protein electrophoresis w a s carried out in gels of
polyacrylamide with reduced condition and in the presence o f SDS (sodium dodecylsulphate) (Laemli, 1970).
T h e gels w e r e discontinous with a first stacking gel
(4 % o f acrylamide) and a s e c o n d with 10 % of acrylamide.
T h e samples loaded w a s mixed with equal amounts
o f the antigenic solution and sample buffer, and then
heated in a bath o f boiling water for 4 min. T h e e l e c trophoresis was carried out at 4° C and 200 V for 45 min
and the separated proteins w e r e transferred to nitrocellulose m e m b r a n e s ( B i o - R a d ) of 0.22 pm ( T o w b i n et
al., 1 9 7 9 ) . T h e transfer lasted 12 h at 4 ° C and 30 V,
in a buffer o f 20 % methanol, 25 mM o f Tris, and
192 mM o f glycine, pH 8.3.
220
T h e nitrocellulose m e m b r a n e w a s b l o c k e d for o n e
hour at room temperature, with skimmed p o w d e r e d
milk at 5 % in PBS, and then w a s h e d for five minutes
in PBS-Tween at 0.3 % (PBS-T). T h e membranes placed
on a Mini Protean II Multiscan ( B i o Rad., Richmond,
CA, USA) w e r e incubated with a 1:200 dilution of antiSA or anti-ES o f A. simplex
s.l. sera (primary antibodies). After w a s h e d for three times with PBS-T, the
m e m b r a n e s w e r e incubated with a 1:30,000 dilution o f
alkaline phosphatase conjugated goat anti-rabbit IgG
(Sigma) (secondary antibody) for o n e hour at r o o m
temperature with light stirring. With washings as the
above, antigen bands w e r e visualized with 5 - b r o m o 4 - c h l o r o - 3 - i n d o l y l p h o s p h a t e / n i t r o b l u e tetrazolium
( B I C P - N B T ) (Bio-Rad).
RESULTS
R
abbit anti-SA and anti-ES sera o f A. simplex s.l.
had similar antigen recognition patterns and
numerous bands against the somatic antigens of
A. lumbricoides,
A. suum. T. canis, A. simplex s.l., A. physeteris, H. aduncum
and H. fabri and against the excretory-secretory antigens o f A. simplex s.l. w e r e detected.
T h e s e results are s h o w n in the Table I.
ASSAYS WITH ANTI-SA SERUM OF A. SIMPLEX S.L.
Most o f the shared bands w e r e found b e t w e e n 30 and
> 212 k D a (Fig. 1A). Bands o f approximately 60, 75,
110 kDa and two b a n d s greater than 212 kDa w e r e
observed in the majority of the antigens. However, the
anti-SA serum recognised a 25 kDa band only in SA
of A. simplex s.l. and low Mw b a n d s ( 1 9 and 22 k D a )
in both antigens o f A. simplex s.l. and in the somatic
antigens of A.
physeteris.
ASSAYS WITH ANTI-ES SERUM OF A. SIMPLEX S.L.
T h e results obtained with the different antigens are presented in Fig. I B . This i m m u n e serum r e c o g n i s e s
c o m m o n bands o f 7.2, 36, 75, 110, 212 and > 212 kDa,
A n t i AS
A. simplex
Anti ES
s.l.
A. simplex
serum
serum
A. simplex
s.l. ( A S )
20
23
A. simplex
s.l. ( E S )
19
12
26
A.
lumbricoides
19
A.
suum
13
20
T.
cuius
10
15
18
18
1 1
14
12
14
A.
physeteris
H.
aduncum
H.
fabri
s.l.
T a b l e I. - C o m m o n b a n d s , b e t w e e n n e m a t o d e s p e c i e s , o b t a i n e d b y
immunoblot.
Fig. 1. - Cross-reactivity r e v e a l e d b y i m m u n o b l o t t i n g b e t w e e n Anisakis
w i t h anti-SA s e r u m o f A. simplex
simplex
s.l a n d o t h e r n e m a t o d e s . A ) I m m u n o b l o t o f h e l m i n t h e x t r a c t s
s.l. B ) I m m u n o b l o t o f h e l m i n t h e x t r a c t s with anti-ES s e r u m o f A. simplex
s.l. -SA-; ( l a n e e ) A. simplex
s.l. -ES-; ( l a n e f) A. physeteris;
suum;
( l a n e c ) Toxocara
canis;
terothylacium
( l a n e h ) H. fabri.
A r r o w s s h o w s p e c i e - s p e c i f i c a n d g e n u s - s p e c i f i c b a n d s . T h e m o l e c u l a r w e i g h t m a r k e r s are indi-
aduncum;
( l a n e d ) A. simplex
s.l.: ( l a n e a ) Ascaris
( l a n e b ) A. lumbricoides:
( l a n e g ) Hys-
c a t e d o n t h e left.
in the different species of n e m a t o d e s studied. Also it
revealed a band o f low Mw ( 1 4 k D a ) in A. simplex s.l.,
A. physeteris and Hysterothylacium
antigens, two bands
of < 7.2 and 19 kDa in both antigens of A. simplex s.l.
and a band o f 9 kDa only in the ES antigen o f A. simplex s.l.
DISCUSSION
T
he studies of cross-reactivity by different methods
confirm the large antigenic homology among the
nematodes. In this work, somatic and excretorysecretory antigens of the third stage larvae o f A. simplex s.l. and the somatic antigens of the n e m a t o d e s :
A. physeteris,
H. aduncum.
H. fabri. A.
lumbricoides.
A. suum and T. canis,
have b e e n studied by immunoblotting, using rabbit anti-SA and anti-ES sera of
A. simplex s.l.. A study o f this kind had not b e e n d o n e
with A. physeteris
and H. fabri. Although this study has
b e e n carried out with immunized rabbits sera only, w e
believe that the results obtained can apply to a natural
infection. Thus, sera from m i c e infected with live
larvae and immunized with dead larvae recognized a
similar pattern o f bands in immunoblots of ES and SA
antigen preparations (Iglesias et al., 1993); also, Pascual et al., 1999, demonstrated high, medium and low
molecular weigh bands in the sera o f 12 patients with
gastric anisakiasis. Otherwise, the larvae L3 were maintained o f 7-9 days in vitro cultivation to obtain a
greater concentration o f excretion-secretion antigens
and w e should admit that they were obtained so much
antigens o f L3 as of L4, since L3 moulting at 3-5 days
(Iglesias et al., 1997; Iglesias, 1999; Iglesias et al.
2001).
Cross-reactivity was detected with all the heterologous antigens tested, although the greatest antigenic
similarity was obtained with the somatic antigen of
A. physeteris
that revealed 18 c o m m o n bands (Table I).
In the case of the anti-ES serum, a strong reactivity with
the antigens o f A. suum and A. lumbricoides
( 2 0 and
19 bands, respectively) was also demonstrated.
T h e results obtained with anti-SA and anti-ES sera
demonstrate the existence of common bands in the ascarids of 60, 75, 110 and > 212 kDa. Some of these bands
had b e e n also revealed by other authors (Akao et al.,
1990; D e Baere et al. 1992; Garcia et al. 1997; Carretero et al, 1998; Iglesias et al, 1996; Iglesias. 1998).
However, the high Mw bands (212 and > 212 kDa)
observed by us in the most of the ascarids studied both
b y electrophoresis (unpublished data) and by blotting,
have not b e e n detected by other authors either in
cross-reactivity studies or in sera of patients with invasive anisakiosis, but they have b e e n revealed with the
serum o f patients allergic to Anisakis
(Montoro et al.,
1997; Valero et al., 2 0 0 3 ) .
Specie-specific c o m p o n e n t s have also b e e n revealed:
o n e < 7.2 kDa in the two A. simplex
s.l. antigens, a
b a n d of 9 kDa only in the ES antigen and another o n e
of 25 kDa in the AS antigen (Figs 1A, 1 B ) .
Anti SA and anti ES sera, detect a 19 kDa protein in
A. simplex
s.l. antigens and the anti SA serum also
221
revealed this c o m p o n e n t in the somatic antigen o f
A. physeteris.
Therefore, this protein could b e considered to b e specific o f the Anisakis genus; w e think
that it will b e interesting to look deeply into the study
o f these low Mw antigens in view o f possible u s e in
immunodiagnostic. Other authors also demonstrated
that the specificity is found in the low molecular mass
proteins, s o Hwang et al., 2 0 0 3 , reported a specificantigen in L3ESP, located at less than 13 kDa and recognized b y sera from persons diagnosed with anisakiasis. Also, specific bands with low Mw were detected
in somatic antigens o f Anisakis
(Iglesias et a l . , 1993;
Iglesias et a l . , 1996); A. suum and T. canis (Maizels et
a l . , 1984; Magnaval et a l . , 1991) and in the sera o f
patients allergie to A>iisakis (Del Pozo et al., 1996; Montoro et al., 1997; Carretero et ai, 1998).
Anti-ES serum revealed a protein o f 14 k D a in the
somatic antigens o f A. simplex
s.l., A. physeteris and
H. aduncum
and in the excretory-secretory antigens
o f A. simplex
s.l (Fig. I B ) ; otherwise, t h e anti SA
serum did not detect this protein in any case. T h e fact
that this c o m p o n e n t was only detected in the antigens
o f Anisakis
and Hysterothylacium
and with the ES
serum, s e e m s to indicate that this is released b y the
larvae, into t h e m a i n t e n a n c e m e d i u m in sufficient
amounts, to produce an antibody response in immunized rabbits and that it is present, in a lower c o n c e n tration, in the somatic antigens. This protein, characteristic o f t h e Ascaridoidea, w a s not detected in the
other parasitic n e m a t o d e s supporting the findings o f
Kennedy el ai, 1988 and K e n n e d y et al., 1989, about
the 14 k D a antigen which is produced in different
quantities b y the m e m b e r s o f this superfamily and can
even presents structural differences in the molecule,
that would condition its immunogenicity.
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