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Taeniid cestodes in Tibetan foxes (Vulpes Ferrilata) detected by copro-PCR: Applications and challenges

Tibetan foxes (Vulpes ferrilata) have been confirmed as the main wild definitive hosts in echinococcosis transmission in the eastern Tibetan Plateau. However, little information is available about the epidemiology in wildlife from the perspective of the Taeniidae family, which is essential knowledge in understanding the epidemiology and phylogeography of cestode species in the Tibetan plateau. Therefore, in this study, we used copro-PCR techniques, by amplifying nad1 and cox1 gene fragments, to detect the taeniid species from Tibetan fox feces collected in Shiqu County, (Sichuan Province, China), eastern Tibetan Plateau. Phylogenetic relationships between amplified sequences and existed Taenia species genotypes were evaluated. Then, the maximum prevalence (positive PCR results from at least one primer pair) and the conservative prevalence (positive PCR results from at least two primer pairs) were calculated. Thirty-six Tibetan fox feces were analyzed. Echinococcus multilocularis (conservative prevalence ± 95% CI: 22.2% ± 13.6%; maximum prevalence ± 95% CI: 33.3% ± 15.4%) and E. shiquicus (2.8 ± 5.4%; 8.3 ± 9.0%) was detected. Meanwhile, DNA fragments of T. polyacantha were detected with high similarity to NCBI sequences (cox1, 94.0%) and to the larva sample DNA sequenced in this study (93.4%), and were supported by phylogenetic analysis. Thus, T. polyacantha might infect Tibetan foxes (5.6% ± 7.5%, 11.1% ± 10.3%). Our limited findings in the epidemiology of parasitic Taenia species suggest that sylvatic transmission cycles for a more species-rich Taeniid community must be established between wild canids and small mammals than just for the two Echinococcus species. Besides, discrepancies in different primer pairs in detecting the taeniid species were evaluated. The sensitivity of some widely used universal primer pairs was poor in detecting Taenia species from canid copro-DNA samples. It is still challenging to the development of effective taeniid species-specific molecular markers especially for non-zoonotic species.


Introduction
The family Taeniidae is composed of more than 150 recognized species in genus Taenia, Echinococcus, Hydatigera, and Versteria (Nakao et al., 2013a), in which many species can cause serious parasitic human and animal diseases (see review Hoberg, 2002Hoberg, , 2006. The intestinal infections (taeniasis) caused by the adult stages of Taeniidae cestodes, systemic infections (cysticercosis, coenurosis, echinococcosis) agented by their larvae phase have been uncovered within a range of humans, domestic animals, and wildlife (Eckert et al., 2004;Nakao et al., 2013b;Lavikainen et al., 2014). Understanding the epidemiology and phylogeography of the Taeniidae species is not only vital for understanding the evolutionary mechanisms of cestode species, but also essential in the surveillance and prevention of infectious pathogenic organisms.
The eastern Tibetan Plateau is recognized as a serious, endemic area of human parasitic diseases caused by the Echinococcus and Taenia species (Wang, 2016;Zhou, 2018). The transmission of echinococcosis has been shown to involve a complex wildlife reservoir in which Tibetan foxes (Vulpes ferrilata) are the main wild definitive hosts (Xiao et al., 2008;Jiang et al., 2012;Boufana et al., 2013). Although Taenia species infectious to humans have mainly been reported between humans and domestic animals (Li et al., 2006), fox species have been found to be infected by many Taenia species worldwide. For example, the red fox (Vulpes vulpes) was confirmed to be the host species of T. crassiceps (Rietschel, 1981), T. serialis, T. hydatigena, and T. polycantha (Ziadinov et al., 2010) in various areas of the Eurasian continent, and Karamon et al. (2018) further determined that the infection rate of the Taenia species in red foxes could be 42.5% in central Poland. Regarding the Tibetan plateau, limited information about the epidemiology of taeniid cestodes in wildlife has been reported. For example, Li et al. (2013) and Cui et al. (2015) reported that Tibetan foxes were infected with T. pisiformis, T. crassiceps, T. multiceps, and T. taeniaeformis (Hydatigera taeniaeformis suggested by Nakao et al., 2013a) based on adult worm morphological and molecular identification by hunted individuals post-mortem in Qinghai Province. As an endangered and widely distributed canid species in the Tibetan plateau (Wang and Xie, 2009), general studies addressing the parasitology of Tibetan foxes are usually hampered due to there being limited specimens for analysis.
Non-invading sampling techniques are more proper and effective methods to detect and surveille the prevalence of tapeworms in Tibetan fox populations. For example, molecular identification technologies based on copro-DNA have been used in studies of the Echinococcus spp. prevalence in a Tibetan fox population in the eastern Tibetan plateau (Jiang et al., 2011Yang et al., 2014). Therefore, we tried to establish a general copro-DNA analytic protocol to detect infection of taeniid species in Tibetan foxes based on fecal samples collected in this field study. This approach may be helpful in understanding the under-researched epidemiology and the phylogeography of Taeniidae in wildlife in this region.

Sample collection
We collected Tibetan fox fecal samples from July to August 2012 in Yongbo Valley (32 � 19 0 -34 � 20 0 N, 97 � 20 0 -99 � 15 0 E; elevation, 4300 m), Shiqu County, Ganzi Autonomous Prefecture, Sichuan Province, China. According to the protocol by Jiang et al. (2012), fox feces were collected along line transects. Line transects were designed to start from the bank of the river running through the center of the valley and end at the top of hills on both sides of the valley. The distance between any two lines was more than 500 m to control repeated sampling feces from a same individual (Zuo et al., in composing). Thus, a total of >40 km long line transects were set to cover a 20 km 2 sampling area. Tibetan fox feces were firstly identified by their dark, small, slender shape, and full of small mammal hairs. Feces were stored in 50-ml centrifuge tubes with 75% ethanol separately, and for safety reasons were kept for at least 3 weeks at À 80 � C. Fecal samples needed further tests for copro-DNA quality by checking their origins using Tibetan fox mtDNA analyses before further processing (see 2.2. for details). However, because of the dry and sunny climate in the plateau, fox feces were dried and decomposed quickly in the field. Thus, we classified the freshness of Tibetan fox feces into four levels: fresh, medium, dry, and old (decomposed) (Supplemental Fig. S1). Although Echinococcus DNA could be detected even in a level three (e.g., dry) fecal sample (Wang et al., unpublished data), Tibetan fox mtDNA can only be detected in fresh or medium level fecal samples. Therefore, only fresh and medium fox fecal samples were used in this study.
Adult The pretreatment and copro-DNA extraction followed the protocol in Jiang et al. (2012). Briefly, dissolved and emulsified raw materials from three to four g of each fecal sample were divided evenly into sample A and sample B. Sample A was not treated with a procedure of mechanical disruption (PMD) of taeniid eggshells, while Sample B was. Then, copro-DNA in both Sample A and B would be extracted by QIAamp DNA Stool Mini kit (Qiagen, Hilden, Germany

Copro-PCR
Before taeniid species detection, Sample A of each fecal sample was checked for the Tibetan fox origin using a corpo-PCR protocol developed by Jiang et al. (2011) to detect the Tibetan fox mitochondrial cytochrome b (cytb) gene. Briefly, copro-PCR products using a canid universal primer pair (L14724/H15149; Wayne et al., 1997) were further digested by restriction enzymes BamHI and SspI for Tibetan fox DNA confirmation. This copro-PCR test also worked as a corpo-DNA quality control to exclude fecal samples with poor DNA quality or strong fecal negative conditions inhibiting copro-PCR amplification, both of which can strongly influence the evaluation of the molecular prevalence of taeniid species using Tibetan fox feces . Therefore, only fecal samples confirmed with the existence of the Tibetan fox cytb gene will be used for taeniid species detection.
When detecting taeniid species, Sample B of each fecal sample was used. A total of three pairs of universal primers were used in this study. The first pair of Taeniidae universal primers CO1 JP (JP3/4.5) was selected according to Bowles et al. (1992) to amplify c.440 bp fragments of the mitochondrial cytochrome oxidase subunit 1 (cox1) gene in taeniid species. However, this primer pair is in fact able to amplify DNA from a wide scope of helminth worm species including the Taeniidae family. To restrict the process to Taeniidae species, we designed a new primer pair CO1 ZQ based on CO1 JP. In addition to cox1 gene fragments, ND1 JP (JP11/12) (Gasser et al., 1999) were used to amplify a c. 500 bp region of the mitochondrial NADH dehydrogenase subunit 1 (nad1) gene. Moreover, four pairs of Echinococcus species-specific primers were used to double-check Echinococcus species detecting results by universal primer tests. Because T. asiatica has been detected in fecal samples by the universal primer pair COI ZQ (see Table 2), the species-specific primer pair PHDP2TSAF3 (Gonz� alez et al., 2004) was used to reconfirm the attendance of T. asiatica. All primer pairs information is listed in Table 1.
All amplifications were generated in a 50 μl reaction volume consisted of 25 μl of Premix Ex Taq (Takara Biotechnology, Dalian, China), 1 μl of bovine serum albumin (Takara), 1 μl of each primer pair, 20 μl of ddH 2 O and 2 μl of sample DNA and were performed using a Bio-Rad DNA Engine PTC-200 instrument (Bio-Rad, Hercules, CA, USA). Amplification conditions were as follows: Initial denaturation at 95 � C for 5 min; 35 cycles of denaturation at 95 � C for 30 s; annealing at 52 � C-60 � C for 30 s (Table 1); extension at 72 � C for 45 s; and a final extension step of 72 � C for 10 min. Negative controls were included in each PCR run. All PCR products underwent electrophoresis in 1.5% agarose gels that stained with ethidium bromide (Biotium, Hayward, CA, USA). Gels were developed in UV illumination using a GelDoc-IT™ imaging system (Alpha Innotech Ltd., Staffordshire, U.K.) and the successful amplifications were cut and purified using a TIANgel midi purification kit (Tiangen).

Cloning and sequencing
Target fragments contained in purified products were ligated into a pMD Tm 19-T vector using a pMD Tm 19-T vector cloning kit (Takara), introduced into E.coli DH5α competent cells (Takara) and then cultivated using A-X-I LB medium plates in 37 � C for 8À 12h. The procedure followed the manufacturer's instructions. At least 20 positive colonies were chosen and sequenced for each plate. The sequencing products accomplished by Majorbio (Shanghai) were analyzed using Bio-edit v7.0.9.0 (Hall, 1999) and MEGA7 (Kumar, 2015), and compared with NCBI database (http://www.ncbi.nlm.nih.gov/BLAST).

Definitive host species confirmation
Other than the Echinococcus genus, little data about Taenia species in Tibetan foxes were available. Therefore, when copro-PCR detected Taenia DNA, to confirm if the Tibetan fox is a newly discovered viable definitive host species of this taeniid species, Taenia eggs in the fecal sample were collected according to a modified protocol from Ito (1980) and Mathis et al. (1996). To each target fecal sample, two to five g of feces were dissolved in a 50-ml centrifuge tube with dH2O, passed through a 100 μm sieve to remove large particles, and then centrifuged at 3600 g for 30 min. Sediments were kept and re-suspended by saturated sucrose solution. After centrifuged at 1000 g for 30 min, the supernatant was passed through a 20 μm mesh size sieve. If Taenia eggs (diameter approximately 31-43 μm) existed in the supernatant, they should be retained in the 20 μm sieve, and could be washed out with dH 2 O and checked out under a microscope. To determine which species these collected eggs belonging to, PMD of taeniid eggshells to release the oncosphere was used . Oncosphere DNA was then extracted using the TIANamp Genomic DNA kit (Tiangen) according to the manufacturer's instructions. Then copro-PCR and molecular species identification were carried out using the method explained in section 2.2.1. and 2.2.2.
However, empirical studies suggested that the detecting efficiency of taeniid eggs in field collected fecal samples might be low. For instance, Nonaka et al. (2008) detected 30% of T. taeniaeformis eggs added in dog feces, and Jiang (2012) reported a 40%. Our previous tests suggested that the Echinococcus DNA detecting rate was only 13.3% (16/120 feces) without PMD, and 62% (74/120) with PMD (Jiang, 2012). Taeniid DNA in eggs would be kept longer than tissue materials in Tibetan fox feces by the protection of eggshells. Therefore, disrupting eggshells and releasing egg DNA in fox feces can facilitate a significantly higher taeniid DNA detecting rate. Therefore, to each Taenia DNA detected fecal sample (i.e., Sample B with PMD), we used its Sample A (i.e., without PMD) to detect taeniid DNA again following the taeniid species detection procedure for the Sample B as explained in section 2.2.2. above. We supposed that if the fecal sample did contain taeniid eggs, the copro-PCR result of Sample A should be negative while positive in Sample B.

Phylogenetic analyses
Little data has been reported on Taenia species that infect wildlife in the Tibetan plateau (but see Li et al., 2013;Cut et al., 2015). Thus, if Taenia species cox1 and nad1 gene fragments were detected, maximum likelihood phylogenetic trees (ML trees) were constructed to evaluate the phylogenetic relationships with existing Taenia species. The cox1 and nad1 sequences downloaded from NCBI were aligned and edited with our sequences detected in fecal samples by MEGA 7 (Kumar, 2015). Then, cox1 and nad1 genotypes of taeniid species were calculated by DnaSP 6 (Librado and Rozas, 2009) to build ML trees using all the genotypes from the nine recognized Echinococcus species as outgroups. Modeltest v3.7 (Posada and Crandall, 1998) was used to test for the best-fit models of nucleotide substitution, and ML trees were built using MEGA 7. The robustness of all ML trees was calculated by bootstrapping with 1000 replicates. Detailed information about the sequences and genotypes used in the phylogenetic analyses is provided in Supplemental Table S1.

Statistical analysis
PCR amplification results using different primer pairs can be quite inconsistent because of different amplification efficiency, complexity of copro-DNA background conditions, and other unknown stochastic factors (Jiang et al., 2011. To evaluate the prevalence of a specific taeniid species based on the 36 fecal samples, we established a maximum prevalence by defining a positive sample as at least one positive PCR result from one pair of primers, and the conservative prevalence by at least two positive results from two pairs of primers (Wang et al., 2018). A 95% confidence interval (CI) was used for each prevalence. The χ2 goodness of fit test was used to test whether the detection efficiency was statistically significantly different between primer pairs, especially between universal primer pairs and species-specific primer pairs. All statistics were computed using R 3.5.2 (http://www.r-project.org).

Results
A total of 63 fox feces samples were collected and 36 of them were detected with Tibetan fox cytb DNA thus used for further analyses.

Echinococcus species detection
Both E. multilocularis and E. shiquicus were detected in fecal samples by ND1 JP or CO1 ZQ universal primer pairs and further confirmed by species-specific primer pairs (Table 2). Echinococcus multilocularis DNA sequences were detected from 12 fecal samples, in which cox1 and nad1 sequences were 98.3%-98.8% and 98.1%-100% identical to the two referential E. multilocularis sequences (accession number: NH259774 and MH259778), respectively, from Qinghai, China (Table 2, Supplemental Table S2). The conservative prevalence was 22.2% � 13.6% (�95% CI) and the maximum prevalence was 33.3% � 15.4% (Supplemental Table S3). Regarding E. shiquicus, DNA in only three fecal samples was detected by the cox1 primer pairs as 98.3%-99.5% identical to the referential sequence detected in the Tibetan Plateau of China. The conservative prevalence of E. shiquicus was 2.8 � 5.4%, and the maximum prevalence was 8.3 � 9.0% (Table S3). Mix infection of the two Echinococcus species was only detected in one fecal sample (F12056, Table 2).

Teania species detection
DNA sequences of a Taenia species were amplified in four fecal samples (Sample B) by CO1 ZQ and ND1 JP universal primer pairs. The cox1 sequences amplified using CO1 ZQ showed 93.4% identical to the T. polyacantha larva sample and 94.0% identical to T. polyacantha from Cape Bathurst, Canada (accession number: EU544595), while the nad1 sequences showed 89.0% identical to the larva sample and 89.3% to T. polyacantha from Lappeenranta, Finland (accession number: EU544637) ( Table 2, Table S2). The conservative (5.6% � 7.5%) and maximum (11.1% � 10.3%) prevalence of this T. polyacantha like species were presented in Table S3. In addition, this Taenia species showed 98.8% in cox1 (accession number: KM042890) and 99.1% in nad1 (accession number: KM042889) identical to an unidentified Taenia specimen, Taenia sp. 2 YF-2014, which was collected from a Smokey vole (Neodon fuscus) in Jiuzhi County, Qinghai Province (Fan et al., 2014) (Table S2). Eggs of this Taenia species were not recovered in the four fecal samples, and its DNA was not detected the Sample A of the four fecal samples either.
Meanwhile, cox1 DNA sequences amplified from two fecal samples by the CO1 ZQ primer pair were found to be 99.7% and 99.4% identical to a T. asiatica referential sequence (JQ517298) from Kanchanaburi, Thailand. Comparing with T. asiatica adult worm samples, the two fecal samples both showed 99.4% and 99.0%identical in cox1 sequences Table 2 The results of detecting Taeniidae spp. from Tibetan fox feces using DNA barcoding and specific-primers PCR methods.
( Table 2, Table S2). However, PCR using species-specific primers showed negative results in detecting T. asiatica in the two samples.

Phylogenetic analysis of Taenia species
To further confirm the existence of the Taenia species detected in this study, phylogenetic relationships of their cox1 and nad1 haplotypes with NCBI downloaded haplotypes were calculated and presented using ML trees. When building the ML tree for the cox1 gene, one haplotype of Taenia species (T. polyacantha) and two of T. asiatica were aligned with 55 downloaded haplotypes of Taenia species, and a GTR þ G substitution model was used. Regarding the nad1 gene, one detected Taenia species (T. polyacantha) haplotype and 23 downloaded haplotypes of Taenia species were used to build the ML tree by setting a GTR þ I þ G substitution model. Both the cox1 and nad1 trees revealed that haplotypes of Taenia species (T. polyacantha) detected in this study and downloaded from NCBI belonged to the same T. polyacantha cluster supported by high bootstrap values (99 in the cox1 tree and 100 in the nad1 tree) (Fig. 1). Meanwhile, the two T. asiatica cox1 haplotypes and downloaded T. asiatica haplotypes comprised one cluster with a bootstrap value of 93 ( Fig. 1(a)). The details of the downloaded sequence information is presented in Supplemental Table S1.

Discrepancy of primer pairs in detecting taeniid species
The results of different primer pairs were quite inconsistent in detecting taeniid species (Table 2). The universal primer pair CO1 JP failed to detect any taeniid species but detected many other non-taeniid helminthic species (Table 2), and thus had a significantly lower detection efficiency for taeniid species (χ2 ¼ 15, P < 0.001). The detection efficiency of the other universal and species-specific primer pairs were not statistically significantly different in detecting E. multilocularis (χ2 ¼ 3.714, P ¼ 0.294), E. shiquicus (χ2 ¼ 3.333, P ¼ 0.343), and Taenia species (T. polyacantha) (χ2 ¼ 3, P ¼ 0.223), respectively.

Discussion
Echinococcus spp. are the main taeniid species detected in this study (Table 2). There are three Echinococcus species, E. granulosus, E. multilocularis, and E. shiquicus distributed in the Tibetan plateau (Craig et al., 2019). Echinococccus granulosus and E. multilocularis, two globally distributed taeniid species, cause a potentially lethal zoonosis, Fig. 1. Phylogram of Taenia species, which were found in China, especially in Tibetan Plateau, using the maximum likelihood method generated from partial cox1 (a) and nad1 (b) gene haplotypes. Isolates from Shiqu County were marked with ▴. Sequences from the referential T. polyacantha larva sample collected in Xinyuan County, Xinjiang Autonomous Region, China, shared the same haplotypes with published sequences from Finland marked with ☆. To each sequence, its accession number and geographic region were presented following the taeniid species name and haplotype number. Scale bar was represented in substitutions per nucleotide, and bootstrap values (with 1000 replicates) were marked at the nodes of branches. echinococcosis, threatening more than 50 million people in west China (Wang, 2016). The eastern Tibetan plateau area is one of the most serious endemic regions of the world (Li et al., , 2010. Echinococcus shiquicus is a newly discovered Echinococcus species reported endemic to the eastern Tibetan plateau Craig et al., 2019). Although E. granulosus was confirmed mainly transmitting between cattle and dogs in this area, transmission of E. multilocularis and E. shiquicus involve complex wildlife cycles including small mammals (e. g., rodents and lagomorphs) as intermediate hosts and canids as definitive hosts. Tibetan foxes are the most abundant wild canid species in the Tibetan plateau (Schaller, 1998), and has been regarded as the main wildlife definitive host of E. multilocularis and E. shiquicus Craig et al., 2019). Thus, monitoring the Echinococcus spp. prevalence in Tibetan fox populations provides imperative knowledge of the zoonotic burden in the local wildlife reservoir, which is pivotal to the management and prevention of echinococcosis.
However, although the Tibetan fox is listed as least concern species by IUCN (Harris, 2014), regarding its population in China, habitat deterioration and high pressure of poaching are main threats for a long time. Consequently, the Tibetan fox has been listed as an endangered species by the Chinese Red list since 2005 (Wang and Xie, 2009), which resulted in the limitation on the application of the traditional hunting and post-mortem screening techniques (Qiu et al., 1995) for echinococcosis surveillance purposes. Meanwhile, as a wild canid species, it will always be challenging to catch enough fox individuals for epidemiological studies. Therefore, non-invasive sampling techniques like fox feces sampling and copro-DNA analysis are more convenient and feasible methods to epidemiological studies of taeniid cestodes in wild definitive host species.
Unlike the Echinococcus species, information about the Taenia infection in Tibetan foxes is limited. Taenia polyacantha has a wide distribution from the northern part of the Eurasia continent, from the UK to Japan (Ihama et al., 2000). Several canid species have been confirmed as definitive hosts including the red fox (Vulpes vulpes) (Al-Sabi et al., 2014;Lavikainen, 2008), the arctic fox (Vulpes lagopus) (Stien et al., 2010), the gray wolf (Canis lupus) ( � Cirovi� c et al., 2015), the raccoon dog (Nyctereutes procyonoides) (Thiess et al., 2001), and the domestic dog (C. familiaris) (Umhang et al., 2014). Both cox1 and nad1 DNA fragments were amplified from Tibetan fox fecal samples (Table 2), and haplotypes of these amplicons (including the sequence from the larva sample from Yili, Xinjiang, China) were also grouped with published T. polyacantha sequences with high bootstrap values support in the ML phylogenetic trees (Fig. 1). Meanwhile, although eggs were not isolated from Tibetan fox fecal samples, the copro-PCR results of the four negative A samples and the four positive B samples suggested that T. polyacantha eggs were detected in the four Tibetan fox feces. The procedure of mechanical disruption (PMD) of taeniid eggshells is imperative in detecting DNA in taeniid eggs in Tibetan fox feces. Rausch and Fay (1988a, b) recognized the existence of two subspecies of T. polyacantha, the temperate Eurasian subspecies T. p. polyacantha and the Holarctic tundra subspecies T. p. arctica. The three-division pattern under the T. polyacantha cluster in both cox1 and nad1 trees (Fig. 1) suggested that a more complex subspecies geographic distribution pattern of T. polyacantha may exist. Sequences of cox1 and nad1 from the larva sample collected in Xinyuan County, Xinjiang Autonomous region were highly identical with sequences from Turkey and UK (Table S2), and were grouped to the same genotypes with EU544587 (cox1 tree, Fig. 1(a)) and EU544637 (nad1 tree, Fig. 1(b)) respectively. The Taenia-sp.2-YF-2014 sequences (Table S2) were collected in Jiuzhi County, Qinghai Province (Fan et al., 2014), 275 km away from our sampling site in Shiqu County, Sichuan Province. Molecular data from our samples and Fan et al. (2014) established an eastern Tibetan plateau branch under the T. polyacantha cluster in both cox1 ( Fig. 1(a)) and nad1 trees ( Fig. 1(b)), and had larger differences to haplotypes in other branches of the species (Tables 2 and S2).
Taenia polyacantha is mainly transmitted between canids and small mammals (Boufana et al., 2012). Taenia which is also abundant in our sampling site in Shiqu County (Wang et al., 2018). Therefore, similar to sylvatic transmission cycles of E. multilocularis and E. shiquicus following the food chain composed of Tibetan foxes and small mammals (mainly voles and pikas) Wang et al., 2018), a T. polyacantha transmission cycle may also be established between Tibetan foxes and voles. Meanwhile, the Tibetan fox has been confirmed as the definitive host species for T. crassiceps, T. pisiformis, T. multiceps, and T. taeniaeformis (H. taeniaeformis, as suggested by Nakao et al., 2013) in Qinghai Province, China (Li et al., 2013;Cui et al., 2015). Therefore, including our report of infecting T. polyacantha, Tibetan foxes are known to be the definitive host of at least seven taeniid species, including five Taenia species and two Echinococcus species in the Tibetan Plateau area.
In this study, DNA fragments of T. asiatica cox1 gene were detected in two Tibetan fox fecal samples (Table 2). Our study area is located in western Sichuan Province where the three human Taenia species, T. solium, T. saginata, and T. asiatica (Hoberg, 2002), are co-endemic in local Tibetan communities (Li et al., 2006). Although the ML phylogenetic tree analysis revealed that T. asiatica sequences retrieved in this study grouped well with other T. asiatica sequences from western China ( Fig. 1(a)), we failed to detect other mitochondrial and nuclear DNA markers using species-specific primers. Tibetan foxes can forage and defecate around villages (Vaniscotte et al., 2011). Therefore, T.asiatica sequences detection might be the result of Tibetan foxes' occasional feeding on feces from infected humans and defecating without truly infected, or was simply because of the short DNA fragment compared. No matter how, the T.asiatica instance indicated the importance of species-specific primers and multiple genes cross validation.
Combined with the noninvasive sampling technique, fecal DNA species identification techniques are becoming powerful tools in largescale epidemiological studies of helminthic parasites, especially when screening wild and protected definitive host species. Species-specific DNA markers were developed and widely used in the identification of recognized species in the Echinococcus genus (Hüttner et al., 2009;Knapp et al., 2009;Jiang et al., 2012;Boufana et al., 2013). However, among the roughly 50 species in the Taenia genus (Nakao et al., 2013a), except for species specific methods developed for the three human Taenia species (Anantaphruti et al., 2007;Gonz� alez et al., 2004;Okamoto et al., 2010;Li et al., 2017), DNA barcoding techniques using universal primers for specific gene fragments remain the main molecular species identification methodology (Lavikainen, 2014). However, the discrepancy of the detecting abilities of the same universal primer pairs in different scenarios must emphasized. For example, Galimberti et al. (2012) used the universal primer pair CO1 JP (JP3/4.5) (Bowles et al., 1992) with adult worm tissues to identify 16 Taenia species, while the same primer pair failed to detect any infection of Taenia species when copro-DNA was used (Table 2). Indeed, JP3/4.5 was designed based on Fasciola hepatica (Bowles et al., 1992) and thus has a wide detecting spectrum. The complex composition or the low quality of the Tibetan fox copro-DNA may limit the ability of CO1 JP (JP3/4.5) to detect Taenia species. To fix this issue, we designed a CO1 ZQ primer pair, which is exclusively focused on Taeniidae species. Nevertheless, the discrepancy-detecting results using CO1 ZQ and ND1 JP, respectively, hampered us from ascertaining the true infection burden of the Taenia species. Therefore, to make the copro-DNA species identification techniques more effective, efficient species-specific molecular markers to relevant the Taenia species should be developed.

Declaration of competing interest
The authors declared that they have no conflicts of interest to this work.