A tool for diagnosis of Dicrocoelium dendriticum infection: hatching eggs and
molecular identification of the miracidium
Hilda Sandoval (a), María Yolanda Manga-González (a), José María Castro (b) (*)
(a)
Departamento de Sanidad Animal, Instituto de Ganadería de Montaña,
Consejo Superior de Investigaciones Científicas (CSIC) - Universidad de León
(ULE), 24346 Grulleros, León, Spain.
(b)
Área de Microbiología, Departamento de Biología Molecular, Universidad de
León, 24071 León, Spain
( )
* Tel.: 34-987291508; Fax: 34987291409; e-mail: jm.castro@unileon.es
1
Abstract:
DNA primers were designed from the 18S rRNA sequence from the relevant
digenean trematode Dicrocoelium dendriticum to evaluate a PCR-based diagnostic method of this
parasite from its eggs in faeces of naturally and experimentally infected sheep. In order to get
DNA from D. dendriticum eggs several hatching mechanisms were studied. Successful results
were obtained when the eggs were frozen to -80ºC and/or in liquid nitrogen and then defrosted.
This method allowed the opening of the egg operculum and the liberation of the miracidium. DNA
from D. dendriticum adults and from hatching egg miracidia was obtained and an amplification
single band of 1.95 kb was observed using primers designed for the total 18S rRNA sequence in
both cases as well as when the template DNA was from adults of the closely related parasite
Fasciola hepatica; in addition a single and specific 0.8 kb band was obtained when primers based
on an internal partial 18S rRNA sequence were used. The method showed to be useful not only in
samples coming from adults but in eggs from from gall bladder and faeces as well. F. hepatica
internal 18S rRNA primers were also designed and used as a negative control to prove that the
eggs in faeces came from D. dendriticum and not from F. hepatica. A molecular tool able to detect
a minimum of about 40 D. dendriticum eggs in one of the definitive host faeces has been
developed for the first time and could provide a useful molecular tool to improve the conventional
coprological diagnosis for detecting D. dendriticum eggs.
KEYWORDS: Dicrocoelium dendriticum; hatching eggs, 18S RNA
2
Introduction
Dicrocoelium dendriticum is a hepatic parasite that affects numerous mammal
species, mainly ruminants, throughout the world. This digenean causes
dicroceliosis, a hepatic disease of health and economic significance in ovine and
cattle (definitive hosts) breeding in extensive farming systems (Manga-González
and González-Lanza 2005). The life cycle of D. dendriticum is very complex
because it needs land molluscs and ants as first and second intermediate hosts,
respectively, to complete it (Manga-González et al. 2001). When the adult
parasite, which lives in the bile ducts and gall bladder of mammals (definitive
hosts), is mature it lays embryonated eggs which pass through the intestine and
are eliminated in the faeces of the mammals. The D. dendriticum thick-shelled
operculate eggs are more resistant to low temperatures than high ones (Alzieu
and Ducos de Lahitte 1991; Alunda and Rojo-Vázquez 1983) and naturally only
hatch after ingestion by the appropriate land mollusc species.
Diagnosis of dicroceliosis in live animals is currently based on quantitative
coprological (Campo et al. 2000), immunological (González-Lanza et al. 2000)
and biochemical (Manga-González et al. 2004) techniques. The post-mortem
diagnosis is carried out by counting D. dendriticum worms in the liver and gall
bladder and doing anatomic and immunopathogical studies (Ferreras et al. 2007).
Using conventional coprological techniques has the drawback of not detecting
the parasite prior to maturity and therefore eliminates eggs with the host’s faeces.
Prepatency in lambs is 49-79 days post-infection (p.i.) (Campo et al. 2000). Due
to the fact the egg per gram of faeces (epg) count is carried out on a limited
number of samples, the results obtained are variable with a high percentage of
false negatives, which makes it difficult to evaluate the anthelminthic treatments
(Manga-González et al. 2010). According to our information, D. dendriticum
antigens have also not been detected in faeces, although other parasites, like
3
Fasciola hepatica, have been detected (Martínez-Pérez et al. 2012).
The use of molecular biology techniques in D. dendriticum studies is
scarce. The genetic variability of adult parasites collected from sheep and cattle
has been studied using the random amplified polymorphic DNA (RAPD)
technique (Sandoval et al. 1999; Morozova et al. 2002). Moreover, D. dendriticum
have also been molecularly characterized using partial sequencing of 18S rDNA
and the internal transcribed spacer nuclear (ITS-2) (Otranto et al. 2007). The
characterisation of 28S and ITS-2 of ribosomal DNA from adult D. dendriticum
originating from ruminants has also been done (Maurelli et al. 2007;
Bazsalovicsová et al. 2010). As regards the molecular diagnosis in intermediate
hosts, Heussler et al. (1998) used a DNA probe for the molecular detection of D.
dendriticum in ants of Formica spp. and Lasius spp. Recently, Martinez-Ibeas et
al. (2011) detected larval stages in mollusc and ant intermediate hosts with a
PCR technique using mitochondrial (mtDNA) and ribosomal internal transcribed
spacer (ITS-2) sequences.
The PCR technique has been used to detect Echinococcus multilocularis
(Bretagne et al. 1993) and F. hepatica (Martínez-Pérez et al. 2012) eggs in host
faeces. However, we have no knowledge of any studies in which this technique
has been used to detect D. dendriticum eggs. The lack of papers in this case
could be due to the difficulties involved in hatching their eggs outside of the
mollusc intermediate hosts in order to obtain the D. dendriticum DNA.
Different in vitro hatching procedures have been tested in D. dendriticum
eggs but only a few of them have been useful. Thus, Mitterer (1975a) used the
intestinal juice of the Roman snail Helix pomatia with hatching results which were
dependent on the absence of O2 (exposure to N2) and the presence of bacteria,
since hatching failed when bacteria was absent, probably because they may
consume oxygen and produce carbon dioxide. In addition he induced eggs to
open partially by using solutions of formic acid and caproic acid. Later, the same
4
author (Mitterer 2008) described a special chamber device to confirm that a
prerequisite for successful hatching is the removal of oxygen and the presence of
carbon dioxide. In a similar way Ratcliffe (1968) used reducing conditions
(ascorbic acid solutions gassed with nitrogen-carbon dioxide mixtures) for
hatching. Other different conditions have been historically used without much
success.
Considering the above our objective was to develop a PCR technique for
the specific diagnosis of D. dendriticum eggs in the faeces of infected lambs. We
first had to obtain specific D. dendriticum DNA probes from the RNA ribosomal 18
S gene sequence and then hatch the parasite eggs to obtain the DNA.
Materials and Methods
DNA purification from whole parasites and PCR amplification conditions
Genomic DNA was extracted from adult parasites of D. dendriticum and F.
hepatica isolated from the livers of infected sheep slaughtered in the León
(Spain) slaughterhouse. Once the parasites were extracted, they were washed
three times in PBS (Phosphate buffered saline pH 7.4) and gentamicin (40 mg/l)
at 37ºC, frozen in liquid nitrogen and then stored at -80º C until DNA extraction,
using two well known standard methods (Steindel et al. 1993 and Bretagne et al.
1993).
When needed, genomic DNA from either D. dendriticum or F. hepatica
were used to amplify the 18S rRNA gene by the Polymerase Chain Reaction
(PCR) using primers deduced from highly conserved DNA sequences present at
the extreme ends of the gene previously shown to amplify an approximately 2kb
fragment (table 1) under conditions described by Johnston et al. (1993) for
Schistosoma mansoni. The 18S RNA sequence of D. dendriticum is deposited in
the GenBank database under accession number Y11236.
5
Internal partial sequences of the 18S rRNA gene were amplified by PCR
using specific primers deduced for the diagnosis of D. dendriticum (Dd5, Dd3)
and F. hepatica (Fh5, Fh3, GenBank accession number X53047). Amplification
conditions were: one cycle of (95ºC, 3 min) followed by 30 cycles of (95ºC, 30
sec; 55ºC 30 sec: 72ºC 3 min) and a final cycle of (72ºC 10 min). In all cases 50
ng of template DNA was used. Table 1 includes the sequence of the primers
used and other relevant information.
Egg isolation and purification
The samples of faeces with D. dendriticum eggs came from lambs we had
infected with a dose of 3000 D. dendriticum metacercariae per sheep, extracted
from the abdomens of naturally infected Formica rufibarbis ants (Campo et al.
2000). The D. dendriticum eggs without any impurities were collected from those
eliminated by adult parasites from the gall bladder and kept alive in RPMI liquid.
In vitro hatching conditions
Some treatments described for Schistosoma mansoni like exposure to urea,
sucrose, sodium chloride and glycerol were applied to D. dendriticum according
to Kassim and Gibertson (1976); Romia (1991); Xu et al. (1986) and Tchounwou
et al. (1991). In addition conditions designed for the close relative F. hepatica,
like exposure to undissociated CO2, light and cooling, dark and different pH
values (Mitterer, 1975b) were applied. Some hatching conditions specifically
described for D. dendriticum described by Mitterer et al. (1975a) and Ratcliffe
(1968) were applied too. Finallly, the freeze-thaw method consisted of exposure
to -80 ºC and/or liquid nitrogen for a minimum of half an hour before a thawing at
room temperature.
DNA isolation from D. dendriticum eggs
6
DNA was isolated from pure eggs from gall bladders or eggs from faeces using
different methods. Initially the Bretagne method (Bretagne et al. 1993), or the
optimized method (Monnier et al. 1996) designed for the detection of
Echinococcus multilocularis DNA in fox faeces using DNA amplification, were
used. In addition the method described by Steindel et al. (1993) as such, or with
a slight modification (increase in the number of heating and phenol precipitation
steps), was assayed. Visual microscopic assays were made throughout the
different steps of the procedures to check the existence of possible opening of
the operculum and/or release of the miracidium.
Genomic DNA was extracted from eggs of D. dendriticum (about 1000
eggs) isolated from the gall-bladder of sheep and kept frozen at -80ºC in PBS
and liquid nitrogen. When required, eggs at -80ºC were defrosted and frozen
again at -80ºC, 30 to 60 min before DNA isolation. Eggs from the original
suspension and ten and /or five fold dilutions were assayed to obtain
chromosomal DNA.
DNA isolation from D. dendriticum eggs from sheep excrements infected
with this parasite
Aliquots of one gram of faeces from sheep containing 700 eggs/gram were used
and kept at -80 ºC. A total of 4 ml of washing buffer and glass beads were added
to the samples, stirring well to separate the faeces. Later, the sample was filtered
using a 150 µm wire mesh. The filtrate was placed in a sedimentation cup. In this
procedure, as in those mentioned earlier, the original filtrate and ten fold dilutions
were used to obtain DNA by the Steindel method after a freeze-thaw step. Some
modifications of the procedure are described later. After electrophoresis to test
for the presence of DNA, a PCR analysis was done with the primers and under
7
the conditions described.
In order to relate hatching to the method of preservation of samples,
faeces from different sheep were divided in 1 ml aliquots: one of them was used
to count eggs by the sedimentation technique and McMaster. Samples containing
about 700 eggs/gram were kept at -80ºC or in liquid nitrogen, -20ºC, 70% ethanol
and room temperature in a cup for 2 days. In these experiments original and ten
or five fold dilutions were assayed.
Results
Different approaches were initially tried in order to get DNA from eggs with no
success; in particular it is remarkable that no DNA from pure eggs isolated from
gall bladder or eggs from faeces was obtained when a known extraction method,
the Bretagne (Bretagne et al. 1993), or the optimized method (Monnier et al.
1996) designed for the detection of E. multilocularis DNA in fox faeces using DNA
amplification, was used. The same happened using the Steindel method
(Steindel et al. 1993). Indeed a visual microscopic assay showed no apparent
opening of the operculum and/or release of the miracidium. Therefore, it was
necessary to test several parameters in order for the eggs to hatch and to obtain
DNA from them.
Different in vitro hatching conditions described for the trematodes S.
mansoni and/or F. hepatica, like exposure to urea, sucrose, sodium chloride,
glycerol, undissociated CO2, light and cooling, dark or pH, were unsuccessful.
Hatching conditions described directly for D. dendriticum, like exposure to formic
and caproic acid, use of increasing concentrations of HCl, H2SO4, KOH (at 65ºC
for 30 min), cedar oil and exposure at room temperature for 2 days, among
others, were equally unsuccessful in obtaining hatching and indeed DNA (data
not shown). Surprisingly, only freezing to -80ºC or exposure to liquid nitrogen for
8
30-60 minutes and later defrosting resulted in the operculum opening. This
procedure allowed egg hatching and miracidium liberation, as could be observed
under the microscope (Fig.1). The mean hatching percentage analysing three
different samples was 82% showing this method is a useful determining factor for
hatching. In this way it was possible to get DNA from the eggs.
Genomic DNA isolated from a single F. hepatica or D. dendriticum
parasite, and also from D. dendriticum eggs, corresponding to purified DNA
samples and five fold dilutions from gall bladder, as well as from faeces
containing about 1000 eggs, were used to amplify the 18S rRNA gene by PCR
with primers (forward NS1, reverse AW13, see table 1) to conserved regions at
the extreme ends. A fragment of about 1.95 kb was obtained in samples from
adults of both species. DNA useful for amplification was extracted from eggs only
after a freeze/thaw step (Fig. 2). The volume of the final DNA samples was 50 µl.
It has to be taken into consideration that the amount of template DNA used in
PCR was one fifth of the final volume (10 of 50 µl); therefore, analysis was with
the amount of DNA extracted from the equivalent of 200, 40 and 8 eggs,
respectively. Significantly, no band was seen when samples containing untreated
eggs (freeze/thaw) were used. However, a band was seen with samples
containing DNA coming from 200 and 40 eggs, respectively. Sequencing of the
fragment from D. dendriticum was carried out as previously described (Sandoval
et al. 1999) to confirm identity.
Internal primers of the 18S rRNA were designed, based on the presence
of hypervariable regions in order to differentiate between adults of both parasites
and to confirm previous results. Primers Dd5/Dd3 (table1) specifically amplified
an 817 bp fragment in D. dendriticum (nucleotides 678-1494, GenBank sequence
Y11236). In a similar way, primers Fh5/Fh3 specifically amplified a fragment of
almost the same size (nucleotides 676-1492 on the GenBank sequence X53047)
in F. hepatica. Amplification bands were seen again in samples containing the
9
DNA extracted from 200 and 40 eggs (a light band), respectively (Fig. 3). When
the freeze-thaw step was omitted, no band from eggs was seen in any case.
In nature eggs are found in the faeces; therefore, an evident step was to try to
isolate DNA from stools. Faeces from different sheep were divided in aliquots;
one of them was used to count eggs by the sedimentation technique and
McMaster. Samples kept at -20ºC, 70% ethanol and at room temperature in a
cup for 2 days showed no apparent egg hatching as seen under the microscope;
no visible DNA was seen in agarose gels and no amplification bands were
detected by PCR. Aliquots of the same samples were deposited before lysis for 1
hour at -80ºC. After a freeze/thaw step the amount of total DNA obtained was
relatively similar among samples. Amplification conditions were as already
mentioned for gall bladder eggs. No clear differences were seen as regards the
yield of DNA obtained with the different procedures used, although slightly higher
values were obtained for samples coming from ethanol (amounts oscillated in all
cases between 850-1950 µg/ml). An expected amplification band of 1.95 kb
appeared using primers NS1 and AW13 (data not shown), as well as a 0.8 kb
band using primers Dd5/Dd3 (Fig. 3) confirming previous results. Primers
Dd5/Dd3 did not amplify F. hepatica DNA. In a similar way, primers Fh5/Fh3 did
not amplify D. dendriticum DNA in any case.
To study the possible significance of faeces weight in improving the
diagnosis method, sheep samples containing 0.38, 0.76 and 1.52 grams of
faeces, respectively (1000 eggs/gram), were processed. The same freeze/thaw
method yielded 102.8 µg/ml, 200 µg/ml and 329.2 µg/ml of DNA, respectively.
When PCR analysis was applied with primers NS1/AW13 and Dd5/Dd3, bands of
2 and 0.8 kb were obtained, respectively. Sequencing confirmed that they
corresponded to the gene for the 18S rRNA. The corresponding bands were
seen in the three samples, indicating that under the conditions described
amplification bands are obtained by processing samples containing about 380-
10
1520 eggs. One fifth of the final volume of template DNA was used for
amplification; therefore, the technique was able to detect DNA from about 75
eggs. Some different attempts to detect amplification bands using DNA from less
than this number of eggs were unsuccessful (data not shown).
Discussion
The hatching of eggs represents a critical phase in a parasite’s life cycle. The egg
of D. dendriticum is a thick-shelled operculate one which is embryonated when
laid. They have an operculum and contain a mature miracidium. Shedding of D.
dendriticum eggs with ruminant faeces occurs uninterruptedly throughout the
year, although the highest values are recorded in the cold period, that is, in Spain
at the end of autumn and in winter (Manga-González and González-Lanza 2005;
Manga-González et al. 2010). Experiments have shown that they can stand
temperatures as low as –20ºC to –50ºC (Boray 1985).The number of eggs per
gram of faeces (EPG) correlates in naturally infected ewes with the intensity of
infection (Rojo-Vázquez et al. 1981). This correlation was also found in
experimentally infected lambs (Campo et al. 2000).
The D. dendriticum eggs hatch in vivo only after ingestion by a suitable
land mollusc intermediate host (Manga-González et al. 2010); therefore one
important question has been to study possible hatching mechanisms. Many
attempts have been made to procure hatching using different methods but only a
few with satisfactory results, among them the experiments done by Ractliffe
(1968) are of note. He studied different in vitro factors for hatching in this
trematode. An appropriate pH and strong reducing conditions triggered the
release in D. dendriticum eggs of an enzyme which acts on the opercular cement
of F. hepatica eggs as well as its own. Mitterer (1975a) induced partial egg
hatching with formic acid and caproic acid solutions. In addition the miracidium
11
hatched in O2 free water of eggs that had been nitrogen- or vacuum-dried. Also,
the use of intestinal juice of the Roman snail Helix pomatia gave hatching results
which were dependent on the absence of O2 (exposure to N2). This author
achieved 50% hatching after exposure to 1.2-14 osmols sucrose /1000 ml free
water from eggs. These and other different methods described for hatching in
closely related trematodes were unsuccessful in our hands as already mentioned.
However, a simple freeze/thaw step previous to DNA extraction was useful not
only in order for the eggs to hatch but also to obtain suitable amounts of DNA for
PCR analysis as well.
Diagnosing D. dendriticum infection on the basis of parasite egg count
using classical sedimentation techniques is very long and tedious. In addition the
detection of eggs could be negative since great volumes are normally needed.
We do not know of any papers published on the detection of D. dendriticum
antigens in the faeces of definitive hosts using the ELISA-sandwich technique.
Diagnosis of dicroceliosis in live animals is currently based on techniques:
quantitative coprological (sedimentation and MacMaster), which allow the number
of D. dendriticum eggs per gram (epg) of faeces from infected animals to be
counted (Campo el al. 2000); 2/ immunological for detecting antibodies
(Jithendran et al. 1996; González-Lanza et al. 2000; Sánchez-Andrade et al.
2003), although this indirect immunological test is non-specific (Fagbemi and
Obarisiagbon 1991), also for direct detection of antigens (we do not know of any
papers published on this); 3/ biochemical, which allow hepatic marker enzyme
alterations and other biochemical parameters produced by the parasites to be
shown (Manga-González et al. 2004). Post-mortem diagnosis of the infected
animals is by counting D. dendriticum worms in the liver (Campo et al. 2000) and
also by anatomical-.immunopathological studies (Ferreras et al. 2007).
Knowledge of D. dendriticum is certainly very scarce at molecular level.
Research on the population structure of D. dendriticum has been carried out both
12
within and among host individuals by isoelectric focusing (Campo et al. 1998);
nonetheless, a genetic analysis based on Random Amplified Polymorphic DNA
showed a high intrapopulation variability among specimens collected from sheep
in a relatively small geographical area (Sandoval et al. 1999). On the other hand
molecular approaches for diagnosing infection are restricted to the detection of D.
dendriticum in ants of Formica spp, an intermediate host. Heusler et al. (1998)
used a repetitive sequence present in the genome of D. dendriticum as a probe
in dot-blot or squash-blot analysis to detect metacercariae in ants, which are the
second intermediate host in the parasite’s life cycle. A recent publication
mentions the detection of larval stages in mollusc and ant intermediate hosts by
PCR, using mitochondrial and ribosomal internal transcribed spacer (ITS-2)
sequences (Martinez-Ibeas et al. 2011). However, to our knowledge no attempts
have been made to amplify hatching egg DNA.
The PCR method amplifying a specific segment of DNA frequently used
for diagnosis seems to be a valuable method for determining the prevalence of
this parasite in sheep. It is well known that a PCR protocol to amplify and detect
DNA extracted directly from excrements presents major difficulties, such as the
presence of inhibitors of the reaction. Indeed, we had many difficulties when
amplifying DNA extracted according to described procedures. Our final tip is to
use an effective and easy to use hatching factor: freezing to -80ºC is useful for
improving the diagnosis of the presence of D. dendriticum eggs in faeces. The
specificity of the primers by testing them against DNA extracted from F. hepatica
is reported as well. Hopefully both factors, a hatching mechanism and a PCR
analysis with specific primers will contribute a little to better knowledge of D.
dendriticum, an financially important Digenea parasite.
In conclusion, this paper introduces a method to hatch D. dendriticum eggs by
freeze-thawing and to obtain their DNA as described. Moreover, using these DNA
a PCR-based molecular tool to detect the presence of D. dendriticum eggs in
13
infected animal faeces is developed for the first time. Nevertheless, it would make
sense to try to improve the method to extract and quantify DNA from a single
egg. This should help to not only valuate the precision of the technique but to
evaluate the effect of anthelmintic treatments applied to animals and to avoid
false negatives obtained when egg counts is carried out using the sedimentation
procedure and Mc-Master.
Acknowledgments
We would like to thank Dr. R. Campo as well as C. Espiniella and M.L. Carcedo, members of the
Parasitology Laboratory at the Spanish National Research Council (CSIC) in León (Spain). This
work was supported by the Spanish CICYT (Project No. AGF96-0416) and Castile and León
Autonomy (Proyect No. CSI5/98). H. Sandoval was supported by a Scientist Research Contract
from the CSIC.
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Fig. 1 Photomicrography of hatching egg of Dicrocoelium dendriticum with the miracidium
liberation by the operculum opening. This egg had been previously frozen in liquid nitrogen. Bar
= 10µm.
Fig. 2 PCR amplification of Dicrocoelium dendriticum and/or Fasciola hepatica DNAs isolated
from adults and/or eggs using primers NS1 and AW13. From left to right: 1-4; samples from gall
bladder (1,2) or sheep faeces (3,4) containing Dicrocoelium eggs (samples containing DNA from
200 eggs) treated to get DNA by the Bretagne and/or the Steindel method, respectively; 5,12, MW
markers Lambda DNA digested with HindIII/EcoRI and 1 kb Plus DNA ladder, respectively; 6-8
D. dendriticum DNA from samples containing 8, 40 and 200 eggs from gall bladder, respectively;
9 same as 8 without a freeze/thaw step; 10, F. hepatica; 11, 13, 14; Samples from faeces
conserved for two days at room temperature, -20 ºC and ethanol without a freeze/thaw step; A
single 1.95 kb band is seen in some cases. Sequencing of the fragment was shown to correspond to
the 18S rRNA, as expected.
Fig. 3 PCR amplification of Dicrocoelium dendriticum and Fasciola hepatica DNAs isolated from
adults and/or hatching eggs using primers Dd5/Dd3 or Fh5/Fh3. From left to right: 1, size control,
0.8 kb band; 2-4; D. dendriticum samples from eggs present in faeces (200 eggs) conserved at -20
ºC, ethanol 70% and room temperature for two days, respectively, treated with a freeze-defrost
step using primers Dd5/Dd3; 5-7; different amounts of faeces corresponding to 75, 150 and 300
eggs, respectively; 8-10, DNA from 8 (no band), 40 (a light band) and 200 eggs from gall bladder,
respectively; 11,12,14,15, D. dendriticum adult (11, 14) and eggs (12, 15) using primers Dd5/Dd3
(11,12) and Fh5/Fh3 (14,15), respectively; 13, 16, F. hepatica DNA from an adult using primers
Fh5/Fh3 and Dd5/Dd3, respectively. When present a 0.8 kb single band is seen.
Table 1 Sequences of primer pairs used in this study with genomic DNA preparations from
Dicrocoelium dendriticum and Fasciola hepatica.
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