A Comparison of Loop-Mediated Isothermal Amplification (LAMP) with Other Surveillance Tools for Echinococcus granulosus Diagnosis in Canine Definitive Hosts
et al. (2014) A Comparison of Loop-Mediated Isothermal Amplification (LAMP) with Other Surveillance
Tools for Echinococcus granulosus Diagnosis in Canine Definitive Hosts. PLoS ONE 9(7): e100877. doi:10.1371/journal.pone.0100877
A Comparison of Loop-Mediated Isothermal Amplification (LAMP) with Other Surveillance Tools for Echinococcus granulosus Diagnosis in Canine Definitive Hosts
Xing-Wei Ni 0
Donald P. McManus 0
Zhong-Zi Lou 0
Ji-Fei Yang 0
Hong-Bin Yan 0
Li Li 0
Hong-Min Li 0
Quan-Yuan Liu 0
Chun-Hua Li 0
Wan-Gui Shi 0
Yan-Lei Fan 0
Xu Liu 0
Jin-Zhong Cai 0
Meng-Tong Lei 0
Bao-Quan Fu 0
Yu-Rong Yang 0
Wan-Zhong Jia 0
Jan S Suchodolski, GI Lab, United States of America
0 1 State Key Laboratory of Veterinary Etiological Biology/Key Laboratory of Veterinary Parasitology of Gansu Province/Key Laboratory of Zoonoses of Agriculture Ministry/ Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences , Lanzhou, Gansu Province , P. R. China, 2 Molecular Parasitology Laboratory , Queensland Institute of Medical Research , Brisbane, Queensland , Australia , 3 Gansu Provincial Center for Animal Disease Control and Prevention , Lanzhou, Gansu Province , P. R. China , 4 Qinghai Academy of Animal Science and Veterinary Medicine , Xining, Qinghai Province , P. R. China , 5 Ningxia Medical University , Yinchuan, Ningxia Hui Autonomous Region , P. R. China
Background: Cystic echinococcosis is highly prevalent in northwest China. A cost-effective, easy to operate diagnostic tool with high sensitivity and specificity would greatly facilitate the monitoring of Echinococcus infections in canine definitive hosts. Methods: The primers used in the LAMP assay were based on the mitochondrial nad5 gene of E. granulosus sensu stricto (E. granulosus s.s., or E.g.s.s.) and were designed using Primer Explorer V4 software. The developed LAMP assay was compared with a conventional PCR method, copro-ELISA and microscopy, using the faeces of dogs experimentally infected with E.g.s.s., and field-collected faeces of domestic dogs including 190 from Qinghai province highly endemic for E.g.s.s. and 30 controls from an area in Gansu, where a domestic dog de-worming program was in operation. Results: The positivity rates obtained for the field-collected faecal samples were 12.6%, 1.6% and 2.1% by the LAMP, PCR and copro-ELISA assays, respectively. All samples obtained from the control dogs were negative. Compared with the conventional PCR, the LAMP assay provided 88.8% specificity and 100% sensitivity. The higher sensitivity of the LAMP method was also shown by the fact that it could detect the presence of laboratory challenge dog infections of E. granulsous s.s. four days earlier than the PCR method. Three copro-samples shown positive by the commercial copro-ELISA were all negative by LAMP, PCR and microscopy, which suggests these samples may have originated from another infection rather than E. granulsous s.s., possibly E. shiquicus or E. Canadensis, which is also present in China. Conclusions: We have developed a potentially useful surveillance tool for determining the prevalence of canine E. granulosus s.s. infections in the field. The LAMP assay may lead to a more cost-effective and practicable way of tracking Echinococcus infections in canids, especially when combined with the copro-ELISA.
Funding: This study was financially supported by Gansu Provincial Key Science and Technology Projects (1203NKDA039); Special Fund for Agro-scientific
Research in the Public Interest (Grant No. 201303037; Grant No. 200903036-07), the Peoples Republic of China; Natural Science Foundation of China (Grant
No. 30960339); and National Health and Medical Research Councils (NHMRCs) Project (APP-1009539), Australia. The funders had no role in study design, data
collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
Cystic echinococcosis (CE), caused by Echinococcus granulosus
(E.g.), is of considerable importance from the public health
perspective and also has a significant socio-economic impact.
There are an estimated 23 million cases of human CE [1,2] with
its global burden in disability-adjusted life years (DALYs)
estimated in 2006 to be 285,000 with an annual economic loss
of US$ 194 million . The most recent (2010) DALY measure for
echinococcosis was reported to be 144,000 . The World Health
Organization has included echinococcosis as one of a group of
zoonoses for its 20082015 strategic plan for the control of
neglected tropical diseases (NTDs) , due to its widespread
distribution in both developing and developed countries .
An important feature of the biology of E.g. is the fact that it
comprises a number of intra-specific variants or strains that exhibit
considerable variation at the genetic level . E.g. sensu lato (E.g.s.l.)
comprises previous 10 genotypes (G1 to G10) with the G1 (sheep
strain) genotype being the prototypical species, infecting humans
and livestock extensively . The G1 (named E. granulosus sensu
stricto, E.g.s.s.) and G6 (camel/dog strain, named E. canadensis)
genotypes have been reported within China . The definitive
hosts of E.g. are canids - dogs, dingoes, foxes, wolves and jackals
which harbour the adult stage in the small intestine. Eggs and
gravid proglottides are released periodically in faeces, into the
external environment. Humans and herbivorous animals become
infected with the metacestode (larval stage) of E.g. following the
ingestion of viable eggs. Transmission is completed when the
viscera of infected intermediate host animals (generally slaughtered
livestock) are consumed by canids , which in turn transmit CE
infection to livestock and humans. Consequently, in order to
effectively control the transmission of E.g., efforts should be
directed towards building an effective surveillance system for
identifying infected canine hosts, an important component for
establishing the epidemiological parameters of CE and for
preventing human and livestock infection . However, the
detection of E.g. in canids is difficult as the infection is generally
asymptomatic and the small proglottides spontaneously discharged
in faeces are usually overlooked . Furthermore, routine
copromicroscopy examination cannot differentiate the eggs of E.g. from
other Taenia species  and, although extensively used, purgation
with arecoline compounds and necropsy of the small intestine
[12,13], is laborious and time-consuming and is impractical for
The application of immunological approaches and polymerase
chain reaction (PCR)-based procedures has proven of value in the
detection of E.g. infection in definitive hosts using defined parasite
copro-antigens or DNA sequences , although challenges
remain in terms of sensitivity and specificity . Further, for
routine laboratory diagnosis and surveillance, DNA methods have
a considerable drawback, in that the sensitivity of conventional
PCR can be severely affected by inhibitory factors present in faecal
samples . The expensive facilities and reagents and the
relatively long time required for test completion are additional
disadvantages [15,16]. Accordingly, we have developed an
effective surveillance tool based on loop-mediated isothermal
amplification (LAMP) for the detection of E.g.-positive canine
faecal samples collected in the field. LAMP-based assays [17,18]
are more convenient and affordable than other traditional
surveillance tools and they can lead to the more rapid detection
of infected canines. Rapid, simple, specific and sensitive LAMP
assays, have been described for the coprodetection of E.g.
infections [19,20]. However, there are no reports, to date, of the
use of LAMP as a tool for the detection of E.g. in faecal samples
from naturally infected dogs. Here we describe the development of
an alternative LAMP assay, based on detecting a specific partial
sequence of the mitochondrial nad5 gene of E.g.s.s. in the faeces of
dogs experimentally infected with metacestodes of the E.g.s.s. We
have assessed its value on canine faecal samples collected in the
field and compared its practicability and diagnostic performance
with conventional PCR, ELISA and microscopy approaches.
Materials and Methods
The institutional ethical committee of Lanzhou Veterinary
Research Institute, Chinese Academy of Agricultural Sciences,
Lanzhou, Gansu Province approved the study (Approval No.
LVRIAEC 2010-005). The experiments using dogs were
undertaken under very strict adherence to the institutional and Chinese
national guidelines for animal husbandry. For any locations and
activities in the field studies, no specific permissions were required,
or no endangered/protected species were involved in.
E. granulosus s.s. materials
An E.g.s.s. isolate  [confirmed subsequently by cox1 and nad1
sequencing (data not shown)] was obtained from a large unilocular
hydatid cyst of E.g.s.s. in a sheep liver at an abattoir in Xining city,
Qinghai province in 2010. The hydatid fluid was aspirated into a
500-ml conical flask and protoscoleces were obtained by
sedimentation. The sediment was washed four times with sterilized
phosphate-buffered saline (PBS). The protoscoleces were then
immediately immersed in Dulbeccos Modified Eagle Media
(DMEM) containing 5% (v/v) fetal bovine serum (FBS) at 37uC
and maintained for two hours in order to determine their viability
by observing their movement microscopically. Samples having
95% viable protoscoleces were used for challenging dogs or stored
at 270uC for DNA isolation.
Experimental infection of dogs
Four 6-month old non-pedigree dogs were purchased from a
dog-market in Lanzhou city, Gansu province. They were treated
with praziquantel (10 mg/kg body weight given on three
consecutive days per month) for two months prior to the study
commencement, and kept in individual cages at the experimental
facility, Lanzhou Veterinary Research Institute, to allow them to
adapt to the living environment and diet. After verification that
they were helminth worm-free, by microscopic examination of
their faeces, each dog (average weight 10 kg) received orally about
10,000 viable protoscoleces of the E.g.s.s. isolate with a meal, and
then they were fed a heat-treated meal once daily. Faecal samples
were collected daily and examined carefully for the E.g.
proglottides with macroscopic and stereo microscopic observation
after sufficiently homogenizing, until day 70 post challenge, from
the bottom of individual cages. They were placed into sterilized
50-ml containers with tight fitting lids and stored at 270uC. The
dogs were euthanized to check the small intestine for the
determination of the number of the E.g.s.s. worms on day 76
Field collection of copro-samples from naturally infected
Faecal samples (n = 190) were collected from individual
domestic dogs in two E.g.-endemic areas; Zhiduo county
(N33u3691.080, E96u03950.040 to N33u48931.470, E95u25922.480;
altitude of approximately 4300 m) and Dari county
(N33u43938.240, E99u25950.51 to N33u39945.930, E98u59927.580;
altitude of 4000,4300 m), Qinghai province . Negative control
dog faeces (n = 30) were collected from Tianzhu county
(N37u11913.900, E102u4893.270; altitude of approximately 2900 m),
Gansu province, where mass dog treatment with praziquantel
(10 mg/kg) had been previously carried out monthly for more
than one and a half years. All collected faecal samples were stored at
270uC before use for microscopic examination or for isolation of
genomic DNA and copro-antigens.
Microscopic examination for the presence of taeniid eggs
Faecal samples from all naturally infected dogs were fully
homogenized and then subjected to a conventional saturated
sodium chloride (NaCl) flotation method . Briefly, 2 g faeces
were washed with distilled water and sedimented by centrifugation
at 2,5006g for 10 min. The supernatant was collected, aliquotted
into tubes and stored at 220uC before being used for
coproELISA detection. The sediment was suspended in saturated NaCl
solution and any eggs present were observed by light microscopy
and recorded. All the copro-samples, including those either
eggpositive or egg-negative, were further tested by the LAMP and
Parasite and host DNA extraction
Genomic DNA (g-DNA), obtained from protoscoleces of the G1
isolate using an Axyprep multisource genomic DNA miniprep kit
(Axygen, CA, USA), acted as an E.g.s.s.-positive control to assess
the sensitivity of the LAMP assay. E.g.s.s. genomic DNA isolated
from faecal samples (f-DNA) was obtained from the faeces of the
experimentally infected dogs and faeces from naturally infected
domestic dogs. Parasite DNA (f-DNA) was extracted from the
faecal samples (200 mg) using Axyprep kits and QIAamp DNA
stool mini kits (Qiagen, Germany). Genomic DNA samples
(gDNAs) from E.g.s.l. including G4 (named E. equinus), G6 and G6/
G7 genotypes (named E. canadensis), E. multilocularis, E. shiquicus, T.
hydatigena, T. pisiformis, T. taeniaeformis, T. multiceps and Dipylidium sp.
were used to determine the specificity of the E.g.s.s. LAMP assay.
The T. taeniaeformis g-DNA was provided by Viktor Dyachenko,
Institute for Infectious Diseases and Zoonoses,
Ludwig-Maximilians-University of Munich, Munich, Germany, the E. shiquicus
gDNA was extracted from a cyst collected from a naturally infected
plateau pika (Onchotona curzoniae) from Shiqu (N33u09954.040,
E97u32959.940; altitude of approximately 4500 m) in 2011, and
the g-DNAs of the E. equinus and E. canadensis isolates were
provided by Antonio Varcasia from Dipartimento di Biologia
Animale, Universita` degli Studi di Sassari, Italy. The other cestode
g-DNAs, isolated using Axyprep kits, were obtained from worms
from experimentally infected dogs at Lanzhou Veterinary
Research Institute. In addition, intestinal contents (200 mg) and
negative faecal samples (200 mg) (n-f-DNA) from uninfected dogs
were obtained from newly born pups and the DNAs were
extracted (Axyprep kits) to serve as negative controls. The
concentrations of the DNA samples were measured using a
Nanodrop 2000 spectrophotometer (Thermo Scientific, China).
Conventional PCR assay
A conventional PCR assay was carried out for comparative
purposes. The PCR primers (Eg1f: 59-CAT TAA TGT ATT
TTG TAA AGT TG-39; Eg1r: 59-CAC ATC ATC TTA CAA
TAA CAC C-39) were used to amplify a fragment of the
mitochondrial 12S rRNA gene of E.g. . The PCR reactions
were performed according to Stefanic et al.  as follows: 50 ml
PCR-mixture comprising 10 mM Tris-HCl (pH 9), 50 mM KC1,
2 mM MgCl2, 200 mM of each dNTP, 0.2 mM each primer,
1.25 U Taq polymerase (TaKaRa, Dalian, China) and 1 ml of
DNA sample. The thermal cycling conditions comprised
incubation at 95uC for 4 min; 35 cycles at 94uC for 30 s, 53uC for 30 s
and 72uC for 30 s with a final extension at 72uC for 10 min.
LAMP primers were designed based on the amplification of a
specific sequence within the mt nad5 gene of E.g.s.s. (GenBank
accession no. AF297617 or NC_008075)  using Primer
Explorer V4 software (http://primerexplorer.jp/elamp4.0.0/
index.html). The sequences for the F3 and B3 primers are located
outside of the two other primers (FIP and BIP) in the E.g.s.s.
mtnad5 gene region. Primers were validated using BLAST software
(http://www.ncbi.nlm.nih.gov/BLAST), and their sequences are
listed in Table 1. The LAMP reaction was performed in a 25 ml
volume with 2 ml of target sample, 1.8 ml of primer mix (40 pmol
each of FIP and BIP, 5 pmol each of F3 and B3), 1.0 ml of Bst
DNA polymerase (8 U), 2.5 ml of 106reaction buffer, 0.5 ml of
25 mM dNTPs, 5 ml of 5 M betaine, 1 ml of 100 mM MgSO4,
and 11.2 ml of ddH2O.
To determine the optimal reaction temperature and time, the
reaction mixture was incubated at 60uC, 61uC, 62uC, 63uC, 64uC
and 65uC, respectively, for 30 min, then heated at 80uC for 5 min
to terminate the reaction; then six different reaction time periods
(10, 20, 30, 40, 50 and 60 min) were compared at the optimal
The specificity and sensitivity of the LAMP assay
To verify the specificity of the LAMP assay for detection of
E.g.s.s. DNA, the LAMP primers were tested using g-DNAs from
other E.g.s.l., E. multilocularis, E. shiquicus, T. hydatigena, T. pisiformis,
T. taeniaeformis, T. multiceps, D. caninum, n-f-DNA (faecal samples
from cestode-free dogs) and dog intestinal tissue as negative
controls. To further confirm the specificity of the LAMP
amplifications, the sequences of the LAMP amplicons were
determined using a modification of the method described by
Nkouawa et al . Briefly, the LAMP products were digested at
37uC with EcoRI (TaKaRa, Dalian, China) for four hours. The
digested products, purified using Axyprep DNA Gel Extraction
Kits, were ligated into pMD-18 T vector at 4uC overnight. The
ligation mixtures were used to transform Escherichia coli JM109
cells by incubating for 12 h at 37uC . Single colonies were
cultivated in Luria-Bertani medium (LB) with Amp + at 37uC for
12 h and then were analyzed by PCR using vector primers
(M13F/M13R). Positive colonies with a PCR-amplified fragment
(,200 bp) of E.g.s.s. mt-nad5 were sequenced by Sangon Biotech
Co., Ltd. (Shanghai, China). In order to determine the sensitivity
of the LAMP assay, E.g.s.s. g-DNA was diluted to 10 ng/ml, then
successively diluted 10 times by the addition of 1 ml of a 1/10
dilution of the previous concentration. The same dilution
procedure was also performed on DNA samples from dog faeces
(f-g-DNA) obtained at different days post-E.g.s.s. metacestode
challenge. In addition, the minimum number of eggs detected by
the LAMP assay was determined in the experiments with faeces
spiked with eggs obtained from E.g.s.s. adults collected from one of
the experimentally infected dogs. The eggs were counted, mixed
with faeces from an uninfected dog and the faecal samples were
then frozen until use.
LAMP/PCR analysis of field-obtained dog faecal samples
The 190 field-obtained faecal samples collected from dogs,
which included samples with taeniid eggs present confirmed by
microscopy, were all subjected to the LAMP and PCR assays. The
f-g-DNA extracted from the faeces of one of the dogs
experimentally infected with E.g.s.s. was used as positive control. The LAMP
and PCR products were electrophoresed on a 1.5% (w/v) agarose
gel with ethidium bromide and photographed using a gel
documentation system. Also, the LAMP products were
characterized by visual inspection after the addition of a 1/10 dilution of 1/
10000 concentration SYBR Green I (Invitrogen) to the reaction
Copro-ELISA analysis of field-obtained dog faecal
Two grams of faecal sample were mixed (1:2) with phosphate
buffered saline (pH 7.2) containing 0.3% (v/v) Tween 20 (PBS-T)
in a 15 ml centrifuge tube at room temperature; the tube was
shaken vigorously and the contents allowed to sediment. The
Name Sequence (5 R 3)
Note: The lower case italicized gaattc in the primers FIP and BIP shows the
position of the introduced EcoRI restriction site.
supernatant was collected into a 2 ml screw capped tube, labelled
with a reference number and stored at 220uC until analyzed using
a commercial copro-antigen sandwich ELISA kit (Zhuhai S.E.Z
Haitai Biological Pharmaceuticals Co., Ltd., Zhuhai, China)
according to the manufacturers instructions. One hundred ml of
faecal supernatant in 0.15 M PBS-T was added to the wells of
polystyrene plates that had been coated with specific antibody
prepared against E.g. antigens (antigen components undisclosed by
the makers of the commercial kit) and incubated at 37uC for one
hour. One positive, one negative and three cut-off controls,
provided in the kit, were placed in wells of each plate. The plates
were washed three times (3 6 3 min) with washing buffer (0.05%
PBS-Tween 20); then, 50 ml anti-E.g. specific antibodies
conjugated with horseradish peroxidase (HRP) (provided in the kit) were
added to each well and incubated at 37uC for 30 min. The wells
were washed three times in the washing buffer, 50 ml each of
colour reagent A and B (provided in the kit with
3,39,5,59tetramethylbenzidine, TMB) were added, the solution was
incubated at 37uC for 30 min in the dark and then the reaction
was stopped with 50 ml stop solution. Absorbance values were read
on a Multiscan ELISA reader at 450 nm. The sample-positive
OD-value was used as the average OD-value of the three cut-off
controls. The sensitivity and specificity of the ELISA was
determined using the conventional PCR assay as reference
Differences among the LAMP, PCR and ELISA procedures
and microscopy for assessing the sensitivity of each test were
determined using One-Way ANOVA with post hoc LSD tests and
the Chi-square test using the software package SPSS 11.5 .
Optimal reaction temperature and time for the LAMP
Of a range of temperatures tested, 63uC was chosen as the
optimal reaction temperature (Fig. 1A). The optimal length of time
for the assay reaction, determined using a range from 10 to 60 min
tested at 63uC, indicated 30 to 40 min was optimal (Fig. 1B).
Accordingly, 40 min was chosen for all subsequent assays.
Analysis of the digested LAMP products
The LAMP products demonstrated typical patterns of
ladderlike bands on agar gels, and their EcoRI digestion products were as
expected (Fig. 1C). The LAMP products with the correct target
Experimentally infected dogs
All six chellanged dogs were euthanized to retrieve the dogs
intestines for the confirmation of the E.g.s.s. infections. Four of the
six dogs yielded 420, 321, 302 and 119 E.g.s.s. worms; no worms
were recovered from the other two dogs.
The specificity and sensitivity of the LAMP assay
E.g.s.s. g-DNAs and f-g-DNA, as well as g-DNAs extracted from
the other parasites and dog host intestinal tissues were tested to
determine the specificity of the LAMP assay for E.g.s.s. DNA. Only
the target gene fragments in E.g.s.s. g-DNA and f-g-DNA
produced the amplified products, (Fig. 1D). The LAMP assay
was 100 times more sensitive (P,0.001) than the PCR, the
detectable level with the former being10 pg gDNA compared with
1 ng with the latter. Similar levels of sensitivity were evident using
f-DNA (data not shown). Further evidence that the LAMP assay
was more sensitive than the PCR was revealed by the fact a
positive result was obtained on average on day 22 post-challenge
infection in the faecal samples of four dogs using the LAMP assay
whereas a positive result was not obtained on average until day 26
post infection using the traditional PCR test (Table 2); the time
difference between the two detection assays was statistically
significant different (p,0.05). The LAMP and PCR assays were
substantially more sensitive than microscopy, as faecal eggs were
not detected visually until day 69 post-challenge on average in the
four infected dogs. Furthermore, the copro-ELISA was positive on
average on day 25 post-challenge infection (P,0.01) (Table 2),
indicating that the LAMP assay was most sensitive of the tools
tested. The faecal samples from the four experimentally infected
dogs were shown to be continuously positive by LAMP, PCR,
copro-ELISA and microscopy until the dogs were sacrificed. The
level of detection sensitivity of the LAMP assay was five E.g.s.s.
eggs per gram of faeces determined three times on separate
occasions (Fig. 1E).
Performance of the four diagnositic assays using
coprosamples from dogs collected in the field
Of the 190 Qinghai Province field-collected dog faecal samples,
24 were positive (12.6%) for the target E.g.s.s. DNA by the LAMP
assay, 3 samples were positive (1.6%) by the PCR assay, 4 samples
were copro-ELISA-positive (2.1%) and 3 were positive (1.6%) by
microscopy (Table 3). All 30 negative faecal control samples were
negative by LAMP, PCR, copro-ELISA and microscopy. Overall,
the specificity and sensitivity of the LAMP assay developed for the
E.g.s.s., calculated by the method of Ma et al. , were 88.8%
and 100%, respectively, when the PCR assay was used as
reference (Table 3).
Cystic echinococcosis (CE) is endemic in many parts of China
 with the highest human prevalence (up to 5%) occurring in
Tibet and in Xinjiang and Ningxia Autonomous Regions [27,28].
The disease has received much attention from the Chinese
Ministry of Health as it is recognized as the main zoonosis having
major public health impact on the rural populations of Western
China [29,30]. Despite its relevance to both public and veterinary
public health, it has proved difficult to establish accurate
prevalence profiles for larval E.g. in intermediate hosts and adult
worms infections in definitive hosts in endemic areas worldwide
. This is partly due to poor reliability of the available
diagnostic tests and the high costs of performing these tests under
field conditions [11,31,32]. Domestic dogs are important definitive
hosts responsible for E.g. transmission, due both to the very close
relationship they have with humans and the fact they are very
susceptible to E.g. infection [33,34]. Current control programs for
E.g. are based mainly on the treatment of domestic dogs using the
highly effective drug praziquantel as a strategy for interrupting the
E.g. life cycle [28,35]. However, a sensitive and accurate diagnostic
test for determining the prevalence and intensity of E.g. in dogs for
large scale surveillance to obtain data on the local infection
pressure is urgently required. Several approaches, with varying
levels of success, have been used to develop tools for the
coprodiagnosis of Echinococcus species in canids [14,36]. Among
these, the copro-antigen ELISA can reliably detect heavy dog
infections  which are responsible for the bulk of
environmental contamination. But it has not proved a useful tool
for the detection of light infections , which are also
important for surveillance to determine the impact of control
options, including chemotherapy [11,40,41].
The specificity of two DNA test systems, involving PCR and
LAMP assays, for the E.g.s.s. was evaluated against a variety of
cestode species including the E.g.s.s., E. canadensis, E. equinus and
other helminth parasites regularly found in the intestines of dogs in
the study area. The outcome of the evaluation thus excluded the
possibility that amplification occurred of non-E.g.s.s. DNA present
in the intestinal contents of field collected dog copro-samples.
None of negative controls gave a signal. Only DNA of the E.g.s.s.
was amplified specifically in the PCR and LAMP assays we
developed. The LAMP assay in the current study showed a
sensitivity that was a hundred times greater than conventional
PCR detection. The LAMP system developed in this study has
overcome the inhibitory components present in copro-samples
. In the current LAMP system, signals were obtained from five
E.g.s.s. eggs per gram of faeces. However, even in dogs with
Table 2. Comparison of the earliest day when faecal samples of E. granulosus-experimentally infected dogs tested positive with
LAMP, PCR, Copro-ELISA and microscopy.
Note: a, LAMP versus PCR; b, LAMP versus Copro-ELISA; c, PCR versus Copro-ELISA; d, LAMP, PCR or Copro-ELISA versus microscopy.
* The LAMP assay revealed a significantly higher level of sensitivity than ELISA, the PCR assay or microscopy (P,0.001; Pearson chi-square test).
mature infections, eggs or gravid proglottids are not shed
continuously and are not homogeneously distributed within faeces.
Some copro-samples were positive in the LAMP assay but no eggs
were evident in the faeces by microscopy. These were probably
due to pre-patent infections on the basis of morphological analysis
of the recovered worms. E.g.s.s. has a pre-patent period of
approximately 6 weeks  and a life expectancy of
approximately 10 months to 1 year . Furthermore, three
coprosamples showing positive by the commercial copro-ELISA were all
negative by LAMP, PCR and microscopy, which suggests these
samples may have originated from another infection other than
E.g.s.s. as the LAMP and PCR assays were confirmed as being
specific for detecting the E.g.s.s. in this study. While ELISA for
antigen detection could reveal the wide-range antigens of
Echinococcus spp., such as due to E. shiquicus or E. canadensis
positive, since these species have been also reported previously
either in animals (including dogs) or/and humans in China
[2,44,45]. The actual field performance of copro-ELISA assays,
particularly currently used commercial copro-ELISA kits [39,46],
are uncertain, due to the potential cross-reactivity of antigens from
other Taenia/helminth species [39,46]. Therefore, some kits based
on monoclonal antibodies against E. multilocularis (such as EmA9)
have been used for the identification of E. granulosus infections,
suggesting the shared antigens exist among Echicococcus species
. Sensitive and specific diagnostic assays are very important as
routine surveillance tools, because their application is required as
an aid to monitor intervention strategies aimed at preventing
human echinococcosis. The LAMP assay we have developed that
is capable of identifying the E.g.s.s. meets these routine
monitoring/surveillance requirements. The operation of the assay is
simple and is readily adaptable to field conditions as it can be
performed simply with an affordable heating block or water bath.
Furthermore, the LAMP reaction results in the precipitation of
white magnesium pyrophosphate in the reaction mixture with the
turbidity increasing with DNA concentration which can be
visualized by the naked eye or quantified with an inexpensive
turbidity-meter . Alternatively, the quantity of DNA can be
measured by a colour change when SYBR Green I, a fluorescent
dsDNA intercalating dye , is employed in the LAMP detection
In summary, considering the advantages of rapid amplification,
simple operation and ease of detection, the field-tested LAMP
assay developed for the E.g.s.s. (the dominant Echinococcus species
impacting on both humans and livestock worldwide) provides a
useful tool for routine E.g.s.s. surveillance in wild and domestic
canine definitive hosts so as to aid in the control of E.g.
We thank the veterinarians and all other colleagues at Center for Animal
Disease Prevention and Control of Dari County, Center for Animal
Disease Prevention and Control of Zhiduo County, Qinghai Province and
Center for Animal Disease Prevention and Control of Tianzhu Tibet
Autonomous County, Gansu Province for collection of field faecal samples.
Also, we are particularly grateful to Antonio Varcasia for providing
gDNAs of the E.g.s.l. isolates and to Viktor Dyachenko for the T. taeniaeformis
Conceived and designed the experiments: XWN DPM YRY WZJ.
Performed the experiments: XWN HBY JFY ZZL HML JZC WGS XL
MTL WZJ. Analyzed the data: XWN DPM YRY WZJ. Contributed
reagents/materials/analysis tools: XWN ZZL HBY LL HML QYL CHL
WGS YLF XL MTL JZC BQF YRY WZJ. Wrote the paper: XWN DPM
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