Acta Tropica 127 (2013) 21–24

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Acta Tropica
journal homepage: www.elsevier.com/locate/actatropica

First report of Echinococcus shiquicus in dogs from eastern

Qinghai–Tibet plateau region, China
Belgees Boufana a,∗ , Jiamin Qiu b , Xinwang Chen b , Christine M. Budke c ,
Maiza Campos-Ponce d , Philip S. Craig a
a
Cestode Zoonoses Research Group, School of Environment and Life Sciences, University of Salford, M5 4WT, Greater Manchester, UK
b
Institute of Parasitic Diseases, Sichuan Centre for Disease Control and Prevention, Chengdu, Sichuan, China
c
Department of Veterinary Integrative Biosciences, Texas A&M University, College Station, TX, USA
d
Section of Infectious Diseases, Department of Health Sciences, VU University Amsterdam, The Netherlands

a r t i c l e i n f o a b s t r a c t

Article history: Echinococcus shiquicus was discovered in foxes and pika wildlife hosts in Sichuan Province, China in 2005.
Received 20 July 2011 Faecal samples from dogs collected in a previous echinococcosis purgation survey from Shiqu County,
Received in revised form 15 February 2013 Ganzi Tibetan Autonomous Prefecture (Sichuan) were screened by coproPCR to investigate the possible
Accepted 23 February 2013
occurrence of E. shiquicus. In addition, coproDNA extracted from 8 necropsied Tibetan foxes (Vulpes fer-
Available online xxx
rilata), the natural host of E. shiquicus, were also included. Thirty (6/20) percent of faecal samples from
dogs were positive for E. shiquicus DNA after PCR amplification of a fragment within the ND1 mitochon-
Keywords:
drial gene. Echinococcus shiquicus was confirmed by sequencing in four dogs and 3 of the 6 dogs were
Echinococcus shiquicus
Dogs
concurrently infected with E. multilocularis. These were also verified by sequencing. Faecal samples from
ND1-coproPCR two Tibetan foxes were shown by PCR to harbour both E. multilocularis and E. shiquicus DNA. One of these
China dual E. multilocularis and E. shiquicus infections in a Tibetan fox was confirmed by sequencing.
Tibetan foxes © 2013 Elsevier B.V. All rights reserved.

1. Introduction small mammal species (including Microtus spp., Ochotona spp.,

Human cystic and alveolar echinococcosis caused by the
metacestodes of Echinococcus granulosus (CE) and E. multilocularis
(AE) respectively are highly endemic in the northwestern Provinces
and Autonomous regions of China including the Qinghai–Tibet
plateau of western China. In these areas dogs are known important
definitive hosts for E. granulosus and E. multilocularis (Craig, 2004;
Li et al., 2010). Recently, a third species, E. shiquicus has been shown
to occur sympatrically in the eastern Qinghai–Tibet plateau region
(Xiao et al., 2005). The zoonotic potential (if any) of this species has
not been determined. The life cycles of Echinococcus spp. involve
a range of mammalian definitive and intermediate hosts (Rausch,
1995). In eastern Tibetan areas domestic and stray dogs serve as
definitive hosts for E. granulosus and E. multilocularis (Budke et al.,
2005). Red foxes (Vulpes vulpes) and Tibetan foxes (Vulpes ferri-
lata) may both serve as final hosts for E. multilocularis, while E.
shiquicus adults have to date only been recovered from Tibetan
foxes (Xiao et al., 2006; Jiang et al., 2012). Sheep and yak serve as the
main intermediate hosts for E. granulosus whereas a number of
∗ Corresponding author at: 43 The Crescent, Manchester, M5 4WT, UK.
Tel.: +44 61 295 4563; fax: +44 61 295 5015.
E-mail address: B.Boufana@salford.ac.uk (B. Boufana).
Lepus spp.) have been recorded as intermediate hosts for E. mul-
tilocularis (Xiao et al., 2003, 2004; Craig, 2004). In contrast, the
only known intermediate host for E. shiquicus is the plateau pika
(Ochotona curzoniae) (Xiao et al., 2005; Ma et al., 2012).
In recent years in China dual infections of E. granulosus and E.
multilocularis have been recorded in the dog definitive host (Zhang
et al., 2006; Xiao et al., 2006; Ma et al., 2012), and E. multilocula-
ris and E. shiquicus metacestodes in O. curzoniae intermediate host
(Xiao et al., 2006). Information on dual infection has so far been gen-
erated through the application of molecular techniques for analysis
of DNA derived from adults or metacestodes of Echinoccocus species
(Zhang et al., 2006; Xiao et al., 2006) as opposed to coprodiagnostic
methods to screen faeces of definitive hosts. To date the molecu-
lar assays applied for coprodetection of E. granulosus (Abbasi et al.,
2003) in definitive hosts on the Tibetan plateau have been shown to
cross-react with E. shiquicus DNA (Boufana et al., 2008). Similarly,
PCR assays used in this region for the detection of E. multilocularis
(van der Giessen et al., 1999) produced positive DNA signals when
E. shiquicus was used as template (Boufana, unpublished obser-
vations). These were two of the molecular assays that had been
optimized and then validated for coproDNA detection of E. granu-
losus and E. multilocularis in canid faeces and were the assays of
choice for the Shiqu County canine purgation study carried out in
2001–2003 prior to the description of E. shiquicus (Xiao et al., 2005).

0001-706X/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.actatropica.2013.02.019
22 B. Boufana et al. / Acta Tropica 127 (2013) 21–24

In that survey 12% and 11% of Tibetan dogs were parasitologically

confirmed infected with E. granulosus or E. multilocularis respec-
tively including some dogs with mixed infections (Budke et al.,
2005). A similar situation was reported from this region where a
domestic dog was coproPCR positive for both these species (Xiao
et al., 2006). E. granulosus/E. multilocularis co-infection was also
confirmed by DNA probes in a dog infection and necropsy survey
carried out in Xinjiang (northwest China) (Zhang et al., 2006). Until
recently no coprotest was available for the detection of E. shiquicus
in definitive host(s). Fig. 1. PCR amplification of Echinococcus multilocularis and Echinococcus shiquicus
DNA extracted from faecal samples of naturally infected dogs. Lanes M marker, lane
In view of the potential widespread occurrence of dual
1, E. multilocularis positive control, lane 2, E. multilocularis DNA from dog SS25AF,
Echinococcus species infections in the eastern Tibetan plateau lane 3, negative control; lane 4, E. shiquicus positive control, lanes 5–6, E. shiquicus
region, as well as the possible misdiagnosis of Echinococcus worms DNA from dogs SS25AF and SS17BE respectively.
and the lack of knowledge regarding the life cycle biology of E.
shiquicus, coproDNA panels from a previous dog purgation study
(2001–2003) (Budke et al., 2005) were re-examined. Faecal sam- (Geospiza, Seattle, WA). Comparison with data held in the GenBank
ples from the Tibetan fox (V. ferrilata), the only known definitive database was accomplished through the use of BLAST biosoftware
host for E. shiquicus, were also included. A coproPCR assay ampli- (www.ncbi.nlm.nih.gov/BLAST/). E. shiquicus and E. multilocularis
fying a species-specific fragment within the mitochondrial NADH nucleotide sequences generated here from the same dog (SS25AF)
dehydrogenase subunit 1 (ND1) gene of E. shiquicus and E. mul- were deposited onto GenBank database under accession numbers
tilocularis was applied to dog and fox faecal samples respectively KC819071 and KC819072 respectively.
(Boufana et al., 2013).
3. Results
2. Materials and methods
3.1. Dogs

2.1. Samples and PCR amplification

In order to investigate the occurrence of E. shiquicus in dogs,
we tested dog purge positive E. granulosus and E. multilocularis
Dog faecal samples collected prior to arecoline purgation
coproDNA samples as well as coproDNA extracted from dog faecal
(n = 15), using a faecal loop (n = 1) and from the ground (n = 4)
ground samples previously analyzed by Budke et al. (2005). Data
in Shiqu County, Ganzi Tibetan Autonomous Prefecture, Sichuan
extracted from that investigation for 20 Tibetan dogs is shown in
Province (China) were included in this study. These had previously
Table 1. CoproDNA extracted from 6 (30%) of those dogs gave pos-
been tested using other molecular probes for E. granulosus (Abbasi
itive signals when amplified using the E. shiquicus ND1 primers
et al., 2003) and E. multilocularis (van der Giessen et al., 1999) in a
yielding a diagnostic 442 bp fragment (Table 1). Four of the E.
purgation study carried out in Shiqu County in 2001–2003 (Budke
shiquicus amplifications were confirmed by sequencing giving a
et al., 2005). All samples were from owned dogs except for two
99–100% identity with ND1 gene of E. shiquicus (accession no.
which were from stray dogs. In addition faecal samples collected
AB159137). Three of these dogs were concurrently infected with
per rectum from 8 necropsied Tibetan foxes (V. ferrilata) para-
E. multilocularis producing 207 bp diagnostic ND1 fragments which
sitologically confirmed, naturally infected with E. shiquicus were
were confirmed by sequencing with 100% identity (Accession no.
included. CoproDNA was extracted using the QIAamp DNA Stool
EU704124) (Table 1). Representative coproDNA amplification of
Mini Kit (Qiagin, Hilden, Germany) according to the manufacturer’s
both E. shiquicus and E. multilocularis DNA in the same dog (ID:
instructions and used as template in an E. shiquicus and E. multilo-
SS25AF) is shown in Fig. 1. Three of the E. shiquicus DNA posi-
cularis NADH dehydrogenase subunit 1 (ND1) mitochondrial gene
tive dog samples (ID: SS39F7, SS25AF and SS39C4) confirmed in
PCR as described by Boufana et al. (2013). Limitations on the quan-
this study by sequencing were also positive for E. multilocularis
tity of DNA available for testing meant that some dog coproDNA
DNA in the initial purgation study using the van der Giessen assay
was not tested using the E. multilocularis ND1 primers (Table 1).
(van der Giessen et al., 1999) and also by the ND1 primers in this
Ethanol precipitation of PCR negative samples was carried out as
study. In view of the observations of cross-reactions of the ‘van
described by Boufana et al. (2013). Twenty microlitres of the ampli-
der Giessen’ protocol with E. shiquicus DNA (Boufana, unpublished
fied PCR product were resolved on a 1.5% (w/v) agarose gel (Bioline,
observations), the infection of these dogs with E. shiquicus was not
UK) in 1× Tris–Borate–EDTA buffer (Severn Biotech, UK) at 110 V,
identified in the initial study of Budke et al. (2005).
stained with gel red DNA dye (Cambridge BioSciences, UK) and
One of the dogs (ID: SS17BE) confirmed by sequencing in the
visualized using Syngene G:Box gel documentation system (Gene-
current study to be positive for E. shiquicus DNA was diagnosed in
Flow, Cambridge, UK). All PCR procedures were carried out in fully
the initial survey as being infected with E. granulosus based on PCR
equipped molecular laboratories following stringent procedures
testing using the method of Abbasi et al. (2003). However, no purge
to ensure the exclusion of contamination. Negative controls (PCR
sample was available for this particular dog. Although this dog may
grade water) were included in all experiments.
have been infected with E. granulosus, the current study has shown
that it was additionally positive for E. shiquicus DNA. Echinococcus
2.2. Sequencing of coproPCR products shiquicus could not be molecularly identified in the previous study
(Budke et al., 2005) as the PCR primers used at the time (after Abbasi
Some of the coproDNA products amplified using the E. shiquiucs et al., 2003) cross-reacted with E. shiquicus DNA (Boufana et al.,
(n = 6) and E. multilocularis (n = 5) probes used in this study 2008). Four Tibetan dogs (ID: SS17AA, SS17AB, SS17AE and SS25DB)
were verified by sequencing. CoproDNA fragments were run which had previously tested positive for E. granulosus using the
on 1× TAE gel and purified using PurLinkTM quick gel purifi- Abassi primers (Abbasi et al., 2003) and were negative for E. mul-
cation Kit (Invitrogen, Paisley, UK) according to manufacturer’s tilocularis DNA based on the PCR of van der Giessen et al. (1999),
instructions. Purified DNA was commercially sequenced (Beckman were also negative for E. shiquicus DNA in the current study and
Coulter, Essex, UK) and analyzed using FinchTV software package thus indicated possible monospecific E. granulosus infections. Of
 B. Boufana et al. / Acta Tropica 127 (2013) 21–24 23

Table 1
PCR amplification and confirmation by sequencing of DNA of E. multilocularis and E. shiquicus from faeces of dogs from Shiqu County (Sichuan Province, China).

Dog ID Site Sample Initial survey resultsa This study

Worm burdens PCR ND1 PCR Sequencing

b c
Eg Em Abbasi van der Giessen Em Es Em Es

SS39F7 Odoma (Tuan Jie 2) Ground – – N P P P P P

SS25AF Yi Niu 1 Purged 0 10,000 N P P P P P
SS39C4 Daroma Village (Ta Yong 1) Purged 0 261 N P P P P P
SS17BE Twanji Ground – – P N N/D P N/D P
SS39B7 Ben Ri (Tent) Purged 0 800 N P P P P N/D
SS34FG Jie Fang 2 Purged 0 1 P P N/D P N/D N/D
SS17AA Twanji Loop – – P N N/D N N/D N/D
SS17AB Twanji Ground – – P N N/D N N/D N/D
SS17AE Twanji Ground – – P N N/D N N/D N/D
SS25DB Yi Niu 1 Purged 0 0 P N N/D N N/D N/D
SS25D4 Xing Rong 1 Purged 140 0 P N N N N/D N/D
SS39AV Yi Niu 1 (Town) Purged 283 0 P N N/D N N/D N/D
SS34EV Ben Ri 2 Purged 0 114 N P P N N/D N/D
SS34EZ Ben Ri 2 Purged 0 5000 N P P N N/D N/D
SS25C7 Yi Niu 2 Purged 0 0 N N N/D N N/D N/D
SS25BU Jie Fang 1 Purged 0 0 N N N/D N N/D N/D
SS25EH Xing Rong 2 Purged 0 0 N P N/D N N/D N/D
SS34BX Xiasha 3 (Town) Purged 0 0 N P N/D N N/D N/D
SS34DQ Azi 1 Purged 0 0 N P N N N/D N/D
SS25CM Ben Ri 3 Purged 0 0 N N N/D N N/D N/D

P, positive; N, negative; N/D, not done.

a
Data extracted from Budke et al. (2005); Eg, Echinococcus granulosus, Em, E. multilocularis, Es, E. shiquicus.
b
Abbasi et al. (2003).
c
van der Giessen et al. (1999).

these four, two were stray dogs (SS17AB, SS17AE). Two purged
dogs (ID: SS25D4 and SS39AV) with E. granulosus worm burdens
of 140 and 283 respectively, that were DNA positive for E. granu-
losus using Abbasi et al. (2003) tandem repeat PCR in the initial
study, were coproPCR negative for E. shiquicus here. This confirms
the coprospecifcity of the ND1 E. shiquicus primers against DNA of
E. granulosus (Boufana et al., 2013).
A further 2 dogs (ID: SS34EV and SS34EZ) with post-purge E.
multilocularis burdens of 114 and 5000 worms respectively, which
had tested positive for E. multilocularis DNA using the PCR of van
der Giessen et al. (1999) and also by the E. multilocularis ND1
primers in the current study, were negative for E. shiquicus. Simi-
larly, three purged dogs (ID: SS25EH, SS34BX and SS34DQ) negative
for Echinococcus worms on purge examination but which were
PCR positive for E. multilocularis, were negative when tested for
E. shiquicus DNA. These dogs probably represent infections with
only E. multilocularis. Three other purged dogs (ID: SS25C7, SS25BU
and SS25CM) which had zero Echinococcus worm burdens follow-
ing coproparasitological examination, and which were coproPCR
negative by previous molecular probes (Abbasi et al., 2003; van
der Giessen et al., 1999), were also negative for E. shiquicus in the
current study.
3.2. Tibetan foxes
CoproDNA from E. shiquicus necropsy positive Tibetan foxes
(n = 8) was used to check for dual exposure to E. shiquicus and E.
multilocularis. When coproDNA extracted from the 8 foxes was
used in the E. shiquicus ND1 PCR assay the diagnostic 442 bp frag-
ment could be amplified from all foxes. In addition, coproDNA of
2 of these foxes was positive when tested with the E. multilocularis
probes producing the diagnostic 207 bp fragment. The E. shiquicus
DNA amplification for these 2 foxes and one of the E. multilocularis
positive copro-amplifications, were verified after sequencing
to be E. shiquicus (accession no. AB159137) and E. mulilocu-
laris (accession no. EU704124) with a 99% and 100% identity
respectively.
4. Discussion
In the current study, we have confirmed by coproPCR and
sequencing, for the first time the natural exposure of Tibetan dogs to
E. shiquicus as well as the dual occurrence in dogs of E. shiquicus and
E. multilocularis. However, the potential role of dogs as definitive
hosts for E. shiquicus remains to be elucidated as does the zoonotic
potential (if any) of this species. Dual human infections of cystic and
alveolar echinococcosis have been recorded in the Tibetan region
(Qiu et al., 2000; Li et al., 2010). A recent retrospective study that
applied DNA primers to tissue biopsy collected from 68 surgically
confirmed Tibetan echinococcosis patients, including cases from
Shiqu County, showed that 38 and 30 of these patients had E. gra-
nulosus or E. multilocularis respectively; E. shiquicus DNA was not
amplified from any of those human hydatid cysts or lesions (Li et al.,
2008). Similarly, 23 surgical samples from Tibetan patients (South
Qinghai Province) were confirmed molecularly as either E. granu-
losus or E. multilocularis (Ma et al., 2012). The pre-patent period of
E. shiquicus in its presumed natural definitive host, V. ferrilata, is
unknown, but may be similar to or shorter than that of E. multi-
locularis (23–25 dpi). Dogs have been observed to predate on the
plateau pika (O. curzoniae) in this region, and dietary analysis also
confirmed this lagomorph in the faeces of dogs (Wang et al., 2010).
We cannot exclude that the E. shiquicus coproDNA detected in dog
faeces may correspond to dietary derived (for example ingestion of
faeces), discharged worms or rejected pre-patent infections rather
than fully established mature tapeworms. According to previous
reports, Tibetan dogs are known to feed on voles, pikas and hares
which are all known to be intermediate hosts of E. multilocularis in
this region (Schantz et al., 2003; Budke et al., 2005). This reflects
the dual occurrence of infection of dogs with E. shiquicus and E.
multilocularis confirmed in this study.
Using both coproPCR and sequencing we have also shown the
dual infection of Tibetan foxes with E. shiquicus and E. multilocula-
ris. This is probably related to the predation of foxes on microtine
rodents and plateau pikas (Jiang et al., 2012). Ochotona curzoniae
has been shown to naturally harbour cysts of E. shiquicus and E.
multilocularis (Xiao et al., 2006). These authors speculated that such
24 B. Boufana et al. / Acta Tropica 127 (2013) 21–24
dual infection could therefore occur in the fox definitive host (Xiao
et al., 2006) which has been confirmed by Jiang et al. (2012) and in
the current study. The occurrence of dual infection of E. shiquicus
and E. multilocularis in both definitive (fox) and intermediate hosts
(small mammals) has been suggested to be associated with high
parasite prevalence, fox predation habits and susceptibility (Xiao
et al., 2006).
The full life-cycle biology of E. shiquicus remains to be deter-
mined. However, it does appear that this species has evolved within
the predatory–prey relationship of Tibetan foxes and plateau pika,
both endemic mammals (Nakao et al., 2010). Echinococcus shiquicus
has to date not been parasitologically confirmed from any other
mammalian species. We now have evidence based on coproDNA
analysis and sequencing that Tibetan dogs are naturally exposed to
E. shiquicus and may develop early or pre-patent or mature infec-
tion. Experimental infections of dogs with E. shiquicus are required
to investigate the infection biology of this species in both foxes and
dogs.
Acknowledgements
This work was supported by a grant (RO1 TW001565) from the
National Institutes of Health and Natural Science Foundation (USA)
Ecology of Infectious Diseases Program and by a Wellcome Trust
Project (094325/Z/10/Z).
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