Discrimination between E. granulosus sensu stricto, E. multilocularis and E. shiquicus Using a Multiplex PCR Assay
Discrimination between E. granulosus sensu stricto, E. multilocularis and E. shiquicus Using a Multiplex PCR Assay
Cong-Nuan Liu 0 1
Zhong-Zi Lou 0 1
Li Li 0 1
Hong-Bin Yan 0 1
David Blair 0 1
Meng-Tong Lei 0 1
Jin-Zhong Cai 0 1
Yan-Lei Fan 0 1
Jian-Qiu Li 0 1
Bao-Quan Fu 0 1
Yu-Rong Yang 0 1
Donald P. McManus 0 1
Wan-Zhong Jia 0 1
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 , CAAS, Lanzhou , People's Republic of China, 2 School of Marine and Tropical Biology, James Cook University , Queensland , Australia , 3 Qinghai Academy of Animal Science and Veterinary Medicine , Xining, People's Republic of China, 4 Molecular Parasitology Laboratory , Infectious Diseases Division, QIMR Berghofer Medical Research Institute , Brisbane, Queensland , Australia , 5 Ningxia Medical University , Yinchuan, Ningxia Hui Autonomous Region , People's Republic of China
1 Editor: Aaron R. Jex, University of Melbourne , AUSTRALIA
Funding: This study was supported by the following:
WZJ: Gansu Provincial Key Science and Technology
Projects (Grant No. 1203NKDA039, http://www.gsstc.
gov.cn/), ZZL: the Special Fund for Agro-scientific
Research in the Public Interest (Grant No.
201303037, http://www.moa.gov.cn), WZJ: the
Special Fund for Agro-scientific Research in the
Public Interest (Grant No. 200903036-07, http://www.
moa.gov.cn), WZJ: the Science Fund for Creative
Research Groups of Gansu Province (Grant No.
Infections of Echinococcus granulosus sensu stricto (s.s), E. multilocularis and E. shiquicus
are commonly found co-endemic on the Qinghai-Tibet plateau, China, and an efficient tool
is needed to facilitate the detection of infected hosts and for species identification.
A single-tube multiplex PCR assay was established to differentiate the Echinococcus
species responsible for infections in intermediate and definitive hosts. Primers specific for E.
granulosus, E. multilocularis and E. shiquicus were designed based on sequences of the
mitochondrial NADH dehydrogenase subunit 1 (nad1), NADH dehydrogenase subunit 5
(nad5) and cytochrome c oxidase subunit 1 (cox1) genes, respectively. This multiplex PCR
accurately detected Echinococcus DNA without generating nonspecific reaction products.
PCR products were of the expected sizes of 219 (nad1), 584 (nad5) and 471 (cox1) bp.
Furthermore, the multiplex PCR enabled diagnosis of multiple infections using DNA of
protoscoleces and copro-DNA extracted from fecal samples of canine hosts. Specificity of the
multiplex PCR was 100% when evaluated using DNA isolated from other cestodes.
Sensitivity thresholds were determined for DNA from protoscoleces and from worm eggs, and
were calculated as 20 pg of DNA for E. granulosus and E. shiquicus, 10 pg of DNA for E.
multilocularis, 2 eggs for E. granulosus, and 1 egg for E. multilocularis. Positive results with
copro-DNA could be obtained at day 17 and day 26 after experimental infection of dogs with
larval E. multilocularis and E. granulosus, respectively.
1210RJIA006, http://www.gsstc.gov.cn/), YRY:
Natural Science Foundation of China (Grant No.
30960339, http://www.nsfc.gov.cn/), YRY: National
Health and Medical Research Council (NHMRC)
Project (Grant No. APP-1009539, http://www.nhmrc.
gov.au/). 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.
The multiplex PCR developed in this study is an efficient tool for discriminating E.
granulosus, E. multilocularis and E. shiquicus from each other and from other taeniid cestodes. It
can be used for the detection of canids infected with E. granulosus s.s. and E. multilocularis
using feces collected from these definitive hosts. It can also be used for the identification of
the Echinococcus metacestode larva in intermediate hosts, a stage that often cannot be
identified to species on visual inspection.
The canid adapted intestinal tapeworms, Echinococcus granulosus, E. multilocularis and E.
shuiqucus are well known to be endemic in Northwestern China. The first two species can
cause fatal disease in humans. Although E. shiquicus has not been reported to infect
humans, all three species can be transmitted by dogs. The very close relationship between
dogs and humans can readily lead to human infection. To aid the surveillance and
management of echinococcosis, effective diagnostic approaches are urgently needed. We
developed a single tube multiplex PCR assay for the accurate identification and discrimination
of the three Echinococcus species for use in both clinical diagnosis and epidemiological
In the most recent taxonomic revision, nine species were recognized in the genus Echinococcus
. Of these, the most important and widespread are E. granulosus sensu stricto (genotypes
G1-G3) and E. multilocularis, which cause cystic echinococcosis (CE) and alveolar
echinococcosis (AE), respectively. The former is commonly associated with livestock and human
infections worldwide whereas the latter is primarily found in voles and humans and is
geographically limited to the northern hemisphere . To date, E. granulosus s.s., E. canadensis
(G6), E. multilocularis and E. shiquicus have been identified in China [3–5]. Both E.
multilocularis and E. granulosus s.s. are particularly widespread in western China, including Qinghai,
Ningxia, Gansu, Xinjiang and Sichuan provinces/autonomous regions, and are well known as
major public health and medical threats. Unlike the other species, E. shiquicus has a very
restricted distribution, being reported only from Qinghai Province, China. This species is not
known to cause human echinococcosis. The intermediate hosts are plateau pikas (Ochotona
curzoniae), in which unilocular cysts occur.
For Echinococcus species in general, dogs, wolves, other canids and cats are definitive hosts
in which adult worms cause sub-clinical infections [6–9]. However, larval Echinococcus spp.
can cause morbidity and mortality in their intermediate hosts which include cattle, sheep,
small mammals (including rodents, plateau pikas, etc.) and humans [10, 11]. It can be difficult
to discriminate morphologically adults of some Echinococcus species, such as E. multilocularis
and E. shiquicus .
To replace traditional morphological methods, a number of molecular approaches targeting
parasite DNA have been developed for identification/discrimination of different life stages of
Echinococcus species in definitive and intermediate hosts [13–15]. Multiplex PCR approaches,
simultaneously using multiple specific primers in a single tube and detecting more than one
target species, are material- and time-saving, precise, efficient and cost-effective when DNA
from a mixture of pathogens may be present in a sample. This approach is also suitable for
mass-screening of samples that may be generated from epidemiological investigations in
endemic areas. Several multiplex PCR methods have been developed for identifying certain
Echinococcus species, but none for the identification of E. shiquicus [16–17].
Based on interspecific variation in mitochondrial genes of the genus Echinococcus, we
designed a multiplex PCR assay with three pairs of specific primers in a single reaction tube for
rapid identification of E. granulosus s.s., E. multilocularis and E. shiquicus originating from
either intermediate or definitive hosts. Further assessment of the sensitivity and specificity of
the multiplex PCR assay was performed using metacestode DNA and copro-DNA to determine
the reliability and accuracy of the new diagnostic tool developed in this study.
Materials and Methods
Dogs and mice used in this study were handled in strict accordance with good animal practice
according to the Animal Ethics Procedures and Guidelines of the People's Republic of China
(Regulations for Administration of Affairs Concerning Experimental Animals, China, 1988).
No endangered/protected species were involved in this study. The dogs and mice used were
also treated in accordance with the institutional procedures and guidelines for animal
husbandry issued by the Ethics Committee of Lanzhou Veterinary Research Institute, Chinese
Academy of Agricultural Sciences (Approval No. LVRIEC2010-005).
Sampling of Echinococcus material
Adult worms were collected from stray dogs during routine work of the endemic
echinococcosis prevention and control program in Dari County, Qinghai Province, P.R. China. A total of
86 Echinococcus spp. metacestode samples from yaks, sheep, Qinghai voles (Microtus/Neodon
fuscus) and plateau pikas were collected on the Qinghai-Tibet plateau, P.R. China. Ten yak
lungs and 16 sheep livers harboring hydatid cysts were collected from abattoirs in Maqu
County, Gansu Province and Xining City, Qinghai Province, respectively. Thirty Qinghai vole
livers and 30 plateau pika lungs harboring hydatid cysts were provided by the epidemic
prevention station of Dari County, Qinghai Province.
Parasite materials were dissected from the host tissue and stored either in 70% ethanol
before molecular analyses, or temporarily stored at 4°C prior to experimental infections of
Experimental infection of dogs
Fifteen dogs (mixed breeds) aged 6–8 months were purchased in Lanzhou City, Gansu
Province, China. These were de-wormed using praziquantel and confirmed to be free of intestinal
parasites by examination of their feces two weeks later. Samples of these feces were retained as
negative controls for the multiplex PCR assay. Live protoscoleces (100,000) of each
Echinococcus spp. were fed independently to five dogs after their viability for dog challenge was
confirmed by microscopy.
Sampling of adults/eggs of Echinococcus spp. from challenged dogs
Dogs were euthanized three months after challenge with protoscoleces. Fecal samples were
collected from the dogs each day prior to sacrifice. After removal of the coarse gut contents, the
small intestine was cut into 15–20 cm lengths and opened to expose the mucosa. Samples,
taken by scraping the mucosa with glass strips, were placed in petri dishes in bio-safety
containers . After addition of a small volume of sterile phosphate-buffered saline (PBS, pH
7.2), the contents were checked for the presence of worms (intact or fragmented) and/or eggs.
Adult worms were removed using a glass needle and washed in PBS three times. All procedures
were performed following appropriate bio-safety conditions .
Fecal sampling from non-experimented definitive hosts
Ten stray dogs, provided by the epidemic prevention station in Dari County, Qinghai Province,
were processed as above to obtain mucosal samples, worms and eggs. Additionally, five fecal
samples from captive foxes were collected from a fur farm in Lanzhou City, Gansu Province.
All the collected fecal samples were frozen at -80°C for at least seven days for bio-safety
reasons. Worm samples were preserved either in 70% ethanol or frozen at (-80°C) in PBS for
DNA samples, extracted from a variety of cestodes (identities confirmed by sequencing and
morphology), were used to determine the specificity of the newly developed multiplex PCR
assay (Table 1). They were kindly provided by the Key Laboratory of Veterinary Parasitology
of Gansu Province, Lanzhou Veterinary Research Institute, CAAS.
DNA extracted from host tissues was used to check for nonspecific reactions or assay
interference that might be caused by contamination of parasite samples with host DNA. Host tissues
DNA extraction from samples
Two hundred mg of each metacestode sample was frozen in liquid nitrogen and ground to
powder after removal of ethanol or PBS by rinsing with ddH2O. Total genomic DNA was
extracted using a QIAGEN DNeasy Blood & Tissue Kit (QIAGEN, Hilden, Germany)
according to the manufacturer’s instructions and stored at -20°C until use.
To minimize the impact of inhibitors on PCR using copro-samples as template, an additional
step of stool flotation in saturated zinc chloride solution was used before copro-DNA extraction
. Briefly, about 20 g (20 ml) fecal material was placed in a 50 ml centrifuge tube, which was
then filled with zinc chloride solution. The tube was vortexed until the fecal material was
completely broken up. The tube was then centrifuged at 1000 ×g for 5 min. Five hundred μl of
the supernatant (usually containing helminth eggs, proglottids or cells of parasites) was
transferred to a 2 ml centrifuge tube, 1.5 ml ddH2O was added to dilute the solution, and the tube
was centrifuged at 12,000 ×g for 10 min. The supernatant was carefully discarded and 200 μl
ddH2O added to suspend the sediment for DNA extraction. Total genomic DNA was extracted
using a QIAGEN QIAamp DNA Stool Mini Kit (QIAGEN, Hilden, Germany) following the
manufacturer’s instructions, and the DNA concentration was determined using a
spectrophotometer (Thermo, NanoDrop 2000, USA) after elution in 50 μl ddH2O for use in the PCR assay.
Genomic DNA was extracted from host tissues using a QIAGEN DNeasy Blood & Tissue
Kit (QIAGEN, Hilden, Germany), according to the manufacturer’s instructions, and stored at
-20°C until use.
The complete mt genomes (mtDNA) of various cestodes (Table 1) available in GenBank
(http://www.ncbi.nlm.nih.gov/) were retrieved to facilitate design of primers specific for E.
granulosus s.s., E. multilocularis and E. shiquicus (Table 1). The sequences were aligned
automatically using Clustal in MEGA5.0 . Primer pairs, expected to be specific for E. granulosus
s.s. (S1 Fig), E. multilocularis (S2 Fig) and E. shiquicus (S3 Fig), were thus obtained. After some
preliminary experimentation, one pair of primers specific for each Echinococcus spp. was
selected for inclusion in the multiplex PCR assay. Sequences of these primers, target genes and
other related information are presented in Table 2.
PCR amplification was carried out in a 25 μl mixture containing 2 μl dNTPs (2.5 mM of each),
2.5 μl 10× ExTaq Buffer (Mg2+ free), 2 μl MgSO4 (25 mM), 0.25 μl ExTaq DNA polymerase
GCTTTAAGTGCGTGACTTTTAATCCC CATCAAAACCAGCACTAATACTCA Amplicon size (bp) Concentration (nM)
(5U/μl) (TaKaRa, Dalian, Liaoning), 100 pg DNA template of each Echinococcus sample, and
all three primer pairs were added according to the final concentrations given in Table 2.
Fragments were amplified using the following optimized thermocycling conditions: 95°C/5 min for
denaturation; 30 cycles of 94°C/30 sec, 55°C/30 sec, 72°C/40 sec; and 72°C/10 min extension.
For all the multiplex PCR assays, positive DNA (DNA templates of the three Echinococcus
spp.) and negative (no-DNA) controls were included.
Identification of PCR products
Amplicons were visualized by electrophoresis in 2.0% (w/v) agarose gels in 1×TAE (40 mM
Tris-acetate, 2 mM EDTA, pH 8.5), stained with ethidium bromide (EB), and viewed under
UV light. The fragments were purified using an agarose Gel DNA Purification Kit (TaKaRa,
Dalian, Liaoning), and then cloned into pMD18-T Simple vectors using a TA cloning strategy.
The recombinant vectors were identified by enzyme digestion and at least two clones for each
DNA region were sequenced by the Shanghai Invitrogen Biotechnology Co. Ltd.
Controls for the multiplex PCR assay
Positive control for fecal sample tests. DNA from protoscoleces of the three Echinococcus
spp. was added to a fecal sample as positive control. Another type of positive control was
provided by the copro-samples that were directly collected from dogs successfully infected with E.
multilocularis or E. granulosus s.s.
Negative controls. To exclude the possibility of contamination in the PCR amplification,
two negative fecal samples (no-DNA) were used. Other negative controls included all reagents
except for the addition of parasite DNA.
Fecal inhibitor controls. To test for potential inhibitors, DNA extracted from
protoscoleces and identified by gene sequencing was added to a negative fecal sample, and subjected to
the multiplex PCR in parallel with the negative fecal sample.
Host tissue controls. DNAs extracted from tissues of dogs and foxes as well as those from
intermediate hosts were tested using the multiplex PCR assay to determine the minimum
contamination level that could cause interference in the assay. Host DNA (0.1, 0.5, 1, 5, 10, 50,
100, 500 or 1000 ng) was mixed into each relevant parasite DNA sample prior to the assay.
Specificity and sensitivity
Specificity. Three pairs of primers were added to each PCR tube with the optimized
multiplex PCR reaction conditions (described above) to test various parasite DNA samples as listed
in Table 1.
Lowest/highest detection limit of DNA using Echinococcus larval tissue. DNA samples
from protoscoleces of the three Echinococcus spp. were quantified by spectrophotometry using
a NanoDrop 2000 (Thermo Scientiific, Wilmington, DE, USA). Serial dilutions of the DNA
template (0.01, 0.02, 0.05, 0.1, 0.5, 1, 5, 10, 50, 100, 500 and 1000 ng) were used to assay the
analytical sensitivity and potential nonspecific amplification of DNA in the multiplex PCR
system. Amplification results were visualized by electrophoresis in a 2.0% (w/v) agarose gel.
Minimum numbers of eggs detectable in fecal samples. One to ten Echinococcus eggs
were added to the diluted negative fecal samples. DNA extracted from these samples was used
in the multiplex reaction to determine the minimum number of eggs that could yield a positive
Earliest time post-infection on which dog fecal samples yielded positive PCR results.
All copro-DNAs, extracted from fecal samples that had been collected every day from
experimentally infected dogs, were tested using the multiplex PCR assay to determine the first
day when a positive signal occurred.
Infections of E. granulosus s.s. and E. multilocularis were successfully achieved in all the
experimentally infected dogs with 5539, 8562, 12535, 18932 and 20775 E. granulosus s.s. and 2893,
3153, 3762, 3864 and 5322 E. multilocularis adult worms being recovered from each group of 5
dogs that were fed with protoscoleces of each species. No adult worms were found in any of the
5 dogs fed larval E. shiquicus. None of the stray dogs was found harboring E. shiquicus or E.
multilocularis; only E. granulosus s.s. adult worms were found in their intestinal contents
(identity confirmed by both morphology and cox1 sequencing). Worm burdens were relatively low
(circa 100–200 worms) in the ten stray dogs examined.
Identification of PCR products
Expected PCR products of 219, 584 and 471 bp were obtained for E. granulosus s.s. (nad1), E.
multilocularis (nad5) and E. shiquicus (cox1), respectively (Fig 1), and products of mixed
templates of the three Echinococcus species are shown in Fig 2. The multiplex PCR products
contained 3 DNA bands (219, 471 and 584 bp) with mixed DNA templates of E. granulosus s.s., E.
multilocularis and E. shiquicus; 2 DNA bands (219 and 584 bp) with E. granulosus s.s. and E.
multilocularis DNA templates; 2 DNA bands (219 and 471 bp) with E. granulosus s.s. and E.
shiquicus DNA templates; and 2 DNA bands (471 and 584 bp) with E. multilocularis and E.
shiquicus DNA templates. DNA sequences of these products corresponded in each case with the
relevant reference sequences in GenBank: E. granulosus (G1) (NC_008075) , E.
multilocularis (NC_000928)  and E. shiquicus (NC_009460) .
Comparison of various sources of DNA. No PCR products were detected when DNAs
from other cestodes (Table 1, labeled with an asterisk) were used in the multiplex assay (Fig 3).
Fig 1. Amplicons of the target genes using the multiplex PCR assay. Lanes 1, 2 and 3, Amplicons of E.
granulosus s.s., E. multilocularis and E. shiquicus respectively; Lane 4, Negative control; M, DNA Marker DL
Fig 2. Amplicons of the mixed templates using the multiplex PCR assay. Lane 1, Amplicons of E.
granulosus s.s., E. multilocularis and E. shiquicus; Lane 2, Amplicons of E. granulosus s.s. and E.
multilocularis, Lane 3, Amplicons of E. granulosus s.s. and E. shiquicus; Lane 4, Amplicons of E. granulosus
s.s., E. multilocularis and E. shiquicus; Lane 5, Negative control; M, DNA Marker DL 2000.
False positive results were never produced from confirmed negative samples. Further, no PCR
products were obtained when DNA samples from various host tissues were used in the
multiplex PCR. Therefore, the specificity of the multiplex PCR for E. granulosus s.s. (G1), E.
multilocularis and E. shiquicus was shown to be 100%.
Copro-DNA templates. Fecal samples, collected from dogs before experimental infection
with Echinococcus spp. and from captive foxes (confirmed parasite-free by microscopy and
DNA analysis), were negative in the multiplex PCR whereas fecal samples from dogs after
experimental infection with either larval E. granulosus s.s. or E. multilocularis were positive.
Furthermore, the PCR products obtained were of the expected sizes, matching those that were
also obtained for all positive controls. No false positive signals were obtained with any negative
Fecal inhibitors and the copro-DNA test. To test for the presence of potential inhibitors,
parallel multiplex PCR assays were performed with positive Echinococcus spp. DNA, negative
fecal samples, and mixtures of Echinococcus spp. DNA and fecal sample DNA as templates.
Positive signals were detectable in the multiplex PCRs with mixtures of the Echinococcus spp.
DNAs and fecal sample DNA as templates. We infer from this that no adverse fecal inhibitors
affected the integrity of the multiplex PCR assay.
Effect of host tissue DNA on the multiplex PCR test. To test for interference due to host
DNA contamination in samples, we added various quantities of host DNA to known quantities
(100 pg) of parasite DNA. Quantities below 500 ng of host tissue DNA (from intestinal,
hepatocyte or pulmonary cells) did not affect PCR outcomes: clear bands of expected sizes were
present in gels. However, smeared bands appeared in the gels if the amount of host DNA
exceeded 500 ng.
Minimum/maximum quantity of Echinococcus metacestode DNA. The lower limit for
the detection of metacestode DNA was 20 pg for E. granulosus s.s., 10 pg for E. multilocularis,
and 20 pg for E. shiquicus, respectively. Clear bands could be visualized up to a maximum
Fig 3. Specificity of the multiplex PCR assay. M, DNA Marker DL 2000; Lanes 1, 6 and 11, Amplicons of E.
granulosus s.s., E. multilocularis and E. shiquicus respectively; Lane 2–5, 7–10 and 12, Other cestode
samples: e.g. E. canadensis (G6), T. hydatigena, T. multiceps, T. pisiformis, T. taeniaeformis, T. solium, D.
caninum, liver tissue of Qinghai vole, lung tissue of plateau pika.
quantity of 500 ng template DNA. Smearing of bands occurred if this amount was exceeded.
Accordingly, the optimum amount of template DNA used was 100 pg for the multiplex PCR as
this quantity produced clear amplicon bands and provided savings on template DNA and PCR
Minimum number of eggs detectable in the fecal samples. Positive PCR products were
obtained in reactions using DNA from as few as two eggs of E. granulosus s.s. and one egg of E.
Earliest time for a positive multiplex PCR assay after experimental infection. Eggs of E.
granulosus s.s. and E. multilocularis were visualized under microscopy at days 47–56 and days
36–44 post-challenge, respectively. The multiplex PCR assay yielded positive results from
copro-DNA 17 days after experimental infection of dogs with larval E. multilocularis and 26
days after infection with larval E. granulosus s.s.
Assessment of feces from stray dogs. Copro-DNA from all the stray dogs infected with E.
granulosus s.s. tested positive in the multiplex PCR.
Fig 4. Sensitivity of the multiplex PCR assay. Lane 1, Amplicons of E. granulosus s.s. and E. multilocularis
with 2 eggs and 1 egg respectively, mixed in fecal sample; Lanes 2 and 3, Amplicons of E. multilocularis from
2 eggs and 1 egg respectively, mixed in fecal sample; Lanes 4 and 5, Amplicon of E. granulosus s.s. amplified
with 2 eggs and 1 egg mixed in fecal sample; Lane 6, Negative control fecal sample; M, DNA Marker DL
China is the most severe pandemic country for cystic echinococcosis (CE), in humans and
livestock, due mainly to E. granulosus s.s., and for alveolar echinococcosis (AE) due to E.
multilocularis in humans and small wild mammals. E. shiquicus is also endemic although it has not been
reported to infect humans. Dual infections of animal hosts with different Echinococcus spp
have been reported in the eastern Qinghai-Tibet plateau region of China [4, 25]. The very close
relationship between dogs and humans can lead readily to human infection. The increasing
number of human AE and CE cases in northwestern China, where considerable numbers of
dogs are present, places a heavy burden on public health and veterinary services. To aid
surveillance, management and diagnosis, effective methods are needed for rapid and accurate
detection and identification of different life cycle stages of the three Echinococcus spp.
simultaneously. The multiplex PCR assay developed in this study provides such a method.
Traditional epidemiological surveys for tapeworms often involve recovery of eggs from
feces of potential definitive hosts. However, morphological identification of Echinoccocus eggs
to species level is practically impossible, prompting the development of several molecular
approaches [26, 27]. Inhibitors present in fecal material that co-purify with parasite DNA
extracted from feces often present a problem for PCR-based methods . In this study, the
QIAGEN QIAamp DNA Stool Mini Kit, containing InhibitEX tablets for removing inhibitors
in fecal samples, was used to purify copro-DNA. The sieving-flotation method was helpful in
overcoming this problem due to its enrichment of worm eggs . The positive control
(protoscolex DNA in fecal samples) used in this study demonstrated the lack of inhibitor effects in
our copro-multiplex PCR assay.
E. granulosus s.s. has been reported as having a pre-patent period of 6 weeks (42 days) [30,
31], while E. multilocularis eggs have been observed in feces at 42–46 days post infection .
However, in the current study we first identified eggs of E. granulosus s.s. at 47–56 days
postchallenge and those of E. multilocularis at 36–44 days post-challenge by microscopy similar to
reports by others [30, 33]. The discrepancies between these studies may be due to the use of
different dog-breeds, ages, nutrient status or the conditions under which the dogs were
maintained. We were unable to experimentally infect dogs with E. shiquicus although the viability of
the challenge sample of protoscoleces was confirmed by microscopy.
PCR-positive signals in this study were obtained from dog fecal samples much earlier (17
days for E. multilocularis and 26 days for E. granulosus) than any other previous studies using
microscopy as a method of detecting infected canid hosts. The much earlier detection of an
Echinococcus infection by the multiplex PCR method compared with egg recovery from feces
and microscopic examination is a marked improvement that can aid surveillance programs
aimed at preventing echinococcosis transmission.
The method developed in this study has achieved high species specificity because it
produced no amplicon from any other helminth (including several that might dual infect with
Echinococcus species in dogs) or from the negative copro-samples (no-DNA). The primer set
(three pairs of primers) multiplex reaction in a single tube worked well with all templates tested
and yielded specific amplicons of the expected length for each of the three Echinococcus spp.
E. granulosus s.s and E. multilocularis are of major public health concern in many endemic
countries globally . A cost effective diagnostic tool is required for echinococcosis
surveillance of definitive and intermediate hosts, and for monitoring the effectiveness of control
programs. The multiplex PCR assay developed in this study provides an effective method that can
be applied in both clinical and epidemiological settings for the identification of Echinococcus
spp in diverse hosts, and would be particularly useful for identifying infected hosts in areas
coendemic for AE and CE.
In this study, we focused on Echinococcus samples collected from the Qinghai-Tibet plateau
region of China, where three species (E. granulosus, E. multilocularis and E. shiquicus) are
known to be endemic. In total, nine species are now recognized in the genus Echinococcus,
including E. granulosus sensu stricto (genotypes G1-G3), E. equinus, E. canadensis (genotypes
G6, G7, G8 and G10), E. ortleppi, E. multilocularis, E. shiquicus, E. vogeli, E. oligarthrus and E.
felidis . None of the three specific pairs of primers developed in this study produced a
PCRamplified product using DNA isolated from E. canadensis (G6 genotype) showing in Fig 3 (the
lane 2 with non-band as a negative result). This is supported by inspection and comparison of
the primer target sequence for the G6 genotype with those of the three Echinococcus spp.,
which showed six base pair differences between them (S1 Fig, S2 Fig and S3 Fig in the
Supporting Supplementary Information). Furthermore, six or more base pair differences are apparent
between the target sequences for E. equinus, E. canadensis (genotypes G7, G8 and G10), E.
ortleppi, E. vogeli, E. oligarthrus and E. felidis. Therefore, it is highly unlikely that any amplicon
would be produced from these species during the multiplex PCR due to its high species
We would like to acknowledge Mr. Naizhi Jiancuo and Ms. Ma Zhuo for their valuable help in
providing tissues of yaks, sheep, Qinghai voles and plateau pikas, and fecal samples from stray
Conceived and designed the experiments: CNL ZZL YRY DPM WZJ. Performed the
experiments: CNL ZZL LL HBY MTL JZC YLF JQL BQF WZJ. Analyzed the data: CNL ZZL DB
YRY DPM WZJ. Contributed reagents/materials/analysis tools: CNL ZZL HBY CNL LL MTL
JZC BQF YRY WZJ. Wrote the paper: CNL ZZL DB YRY DPM WZJ. Collected tissue
materials: MTL JZC YLF JQL.
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