Mitogenomic exploration supports the historical hypothesis of anthropogenic diffusion of a zoonotic parasite Echinococcus multilocularis.

Animal movement across regions owing to human activity can lead to the introduction of pathogens, resulting in disease epidemics with medical and socioeconomic significance. Here, we validated the hypothesis that human activity, such as the transportation of infected animals, has played a significant role in introducing the zoonotic parasite Echinococcus multilocularis into Hokkaido, Japan, by synthesizing and evaluating parasite genetic data in light of historical records. Our analysis indicates that a major genetic group in Hokkaido originated from St. Lawrence Island, USA, which is in accordance with the route suggested by historical descriptions. Moreover, we identified a minor genetic group closely related to parasites found in Sichuan, China. This fact implies that parasite invasion in Japan may result from complex and inadvertent animal translocations. These findings emphasize the anthropogenic impacts on zoonotic parasite spread and provide a crucial perspective for preventing future potential epidemics.

Killing Two Birds with One Stone: Discovery of Dual Inhibitors of Oxygen and Fumarate Respiration in Zoonotic Parasite, Echinococcus multilocularis.

Ascofuranone (AF), a meroterpenoid isolated from various filamentous fungi, including Acremonium egyptiacum, has been reported as a potential lead candidate for drug development against parasites and cancer. In this study, we demonstrated that AF and its derivatives are potent anthelminthic agents, particularly against Echinococcus multilocularis, which is the causative agent of alveolar echinococcosis. We measured the inhibitory activities of AF and its derivatives on the mitochondrial aerobic and anaerobic respiratory systems of E. multilocularis larvae. Several derivatives inhibited complex II (succinate:quinone reductase [SQR]; IC50 = 0.037 to 0.135 µM) and also complex I to III (NADH:cytochrome c reductase; IC50 = 0.008 to 0.401 µM), but not complex I (NADH:quinone reductase), indicating that mitochondrial complexes II and III are the targets. In particular, complex II inhibition in the anaerobic pathway was notable because E. multilocularis employs NADH:fumarate reductase (fumarate respiration), in addition to NADH oxidase (oxygen respiration), resulting in complete shutdown of ATP synthesis by oxidative phosphorylation. A structure-activity relationship study of E. multilocularis complex II revealed that the functional groups of AF are essential for inhibition. Binding mode prediction of AF derivatives to complex II indicated potential hydrophobic and hydrogen bond interactions between AF derivatives and amino acid residues within the quinone binding site. Ex vivo culture assays revealed that AF derivatives progressively reduced the viability of protoscoleces under both aerobic and anaerobic conditions. These findings confirm that AF and its derivatives are the first dual inhibitors of fumarate and oxygen respiration in E. multilocularis and are potential lead compounds in the development of anti-echinococcal drugs.

Data on the combined effect of atovaquone, mefloquine, and 3-bromopyruvic acid against Echinococcus multilocularis protoscoleces.

The dataset presented here is related to a previous research article titled "Mitochondrial Complex III in Larval Stage of Echinococcus multilocularis as a Potential Chemotherapeutic Target and in vivo Efficacy of Atovaquone Against Primary Hydatid Cysts"[1]. In this report, data were collected from aerobic and anaerobic culture assays of E. multilocularis protoscoleces in the presence of three anti-echinococcal drug candidates (atovaquone, mefloquine, and 3-bromopyruvic acid). The data were analyzed for viability of the protoscoleces between day 0 and day 7 upon adding drug candidates. In aerobic condition, all drug candidates caused damage to the protoscoleces, as described previously [1], [2], [3], [4], [5], [6]. Mefloquine, alone as well as in combination with atovaquone, immediately eliminated the protoscoleces, whereas combination of atovaquone with 3-bromopyruvic acid did not show clear synergy. In anaerobic condition, mefloquine, alone as well as in combination with atovaquone, eliminated protoscoleces immediately. 3-Bromopyruvic acid showed stronger efficacy in anaerobic condition than in aerobic condition. Combination of atovaquone with 3-bromopyruvic acid eliminated the protoscoleces, indicating that synergy occurred only under anaerobic condition. The data clarified that combined use of the three drugs eliminated protoscoleces in both aerobic and anaerobic conditions, hence suggesting that these could inhibit aerobic and anaerobic respiration pathways of Echinococcus multilocularis in vivo. The obtained data would be useful for the development of new drug dosing method for alveolar echinococcosis.

Anthelmintic Baiting of Foxes against Echinococcus multilocularis in Small Public Area, Japan.

We distributed anthelmintic baits on a university campus in Japan inhabited by foxes infected with Echinococcus multilocularis to design an effective baiting protocol for small public areas. High-density baiting can reduce the risk for human exposure to the parasite to near zero. However, monthly baiting is recommended to maintain this effect.

Sensitivity comparison between Mini-FLOTAC and conventional techniques for the detection of Echinococcus multilocularis eggs.

Canines serve as the definitive host of Echinococcus multilocularis. This study evaluated the sensitivity of the Mini-FLOTAC technique (MF) for the detection of E. multilocularis eggs in definitive hosts. First, we investigated the effects of heat inactivation and preservative conditions on the detection rate of eggs obtained from experimentally infected dogs. The sensitivity of MF was compared with that of eight other techniques: the centrifugal flotation with sucrose or zinc sulfate, MGL, AMS III, and a combination of MF and flotation/sedimentation techniques. Finally, we compared the sensitivity of MF and the centrifugal flotation with sucrose for the feces of E. multilocularis-infected foxes. The detection rate reached a plateau level with a specific gravity (s.g.) 1.22 for fresh eggs, but the highest rates were obtained with s.g. greater than 1.32 for heat-inactivated eggs. There was no significant difference in the detection rate among the preservative conditions. MF showed significantly higher EPG than the other techniques. Moreover, it showed higher diagnostic sensitivity for the fox feces than the centrifugal flotation technique. These results suggest that heat inactivation may alter s.g. of E. multilocularis eggs and that MF with zinc sulfate (s.g. = 1.32) would be effective for detecting heat-inactivated E. multilocularis eggs.

Early-phase migration dynamics of Echinococcus multilocularis in two mouse strains showing different infection susceptibilities.

The early-phase migration dynamics of Echinococcus multilocularis in the intermediate hosts remain largely unknown. We compared the parasite burden in the intestine, liver and faeces of DBA/2 and C57BL/6 mouse strains using parasite-specific quantitative PCR. Our results indicated that the parasites invaded mainly from the middle segments of the small intestine and completed migration to the liver within 24 h p.i. C57BL/6 mice had lower parasite DNA burdens in the intestine and liver but higher in the faeces than DBA/2 mice, suggesting that parasite invasion of the intestine may be a critical stage regulating susceptibility to E. multilocularis infection in mice.

In vivo efficacy of combination therapy with albendazole and atovaquone against primary hydatid cysts in mice.

Alveolar echinococcosis (AE) is caused by the larval stage of Echinococcus multilocularis. Chemotherapy for AE involves albendazole (ABZ), which has shown insufficient efficacy. More effective chemotherapy for AE is needed. Previously, we have demonstrated that atovaquone (ATV), an antimalarial, inhibits mitochondrial complex III of E. multilocularis and restricts the development of larval cysts in in vivo experiments. Therefore, in this study, we evaluated the efficacy of ABZ and ATV combination therapy on E. multilocularis in culture and in vivo experiments. Protoscoleces were treated with 50 µM ABZ and/or ATV in the medium; the duration of parasite elimination was determined under aerobic and anaerobic culture. In the in vivo experiment, the effects of ABZ and ATV combination treatment in BALB/c mice infected orally with eggs from the feces of an adult-stage E. multilocularis-infected dog were compared with those of standard oral ABZ therapy. In the culture assay, the duration of elimination associated with ABZ and ATV combination treatment was shorter than that associated with ATV alone under aerobic conditions. Protoscolex viability progressively reduced owing to the combination treatment under anaerobic conditions; however, either drug used singly did not exhibit antiparasitic effects under hypoxia. Furthermore, compared with ABZ alone, the combination treatment significantly reduced the growth of the primary cyst in the liver of mice infected orally with parasite eggs (P = .011). ATV enhances the effect of ABZ in the treatment of AE in mice.

Cystic echinococcosis in humans and animals in Egypt: An epidemiological overview.

Cystic echinococcosis (CE), caused by the cestode Echinococcus granulosus (sensu lato), is a serious neglected zoonotic disease in many parts of the world, including Egypt. Thus far, the actual incidence of CE in the Egyptian population remains unknown. Infection with E. granulosus (s.l.) is common among stray dogs in rural and suburban areas owing to the spread of parasite eggs. Herein, we present an updated review of published data on the incidence of CE in humans and animals as well as the genotypes prevalent in Egypt. CE occurs in most parts of Egypt; however, available data are mostly from northern Egypt, particularly Cairo and Giza. In southern Egypt, the disease is likely to be underdiagnosed or underreported. A few risk factors were studied. In the Egyptian population, residency in rural areas, farming, and age were significant factors for acquiring CE. In livestock, age, sex and season have been associated with high prevalence of CE. Several genotypes have been identified among livestock (G1, G4, G5, G6 and G7) and humans (G1, G6 and G7). This literature review underscores the need for a precise national surveillance system to track CE distribution in humans and animals and design appropriate preventive and control strategies for this disease.

Effect of the anti-parasitic compounds pyrvinium pamoate and artemisinin in enzymatic and culture assays: Data on the search for new anti-echinococcal drugs.

The dataset presented herein is related to a previous research article titled "Mitochondrial Complex III in Larval Stage of Echinococcus multilocularis as a Potential Chemotherapeutic Target and in vivo Efficacy of Atovaquone Against Primary Hydatid Cysts" [1]. In this report, data were collected by screening drugs for echinococcosis. We investigated the inhibitory activities of artemisinin and pyrvinium pamoate against the mitochondrial respiratory enzymes in E. multilocularis protoscoleces. Artemisinin did not inhibit mitochondrial complexes I, II, and III. However, pyrvinium pamoate inhibited complex I at 11 µM, although complexes II and III were not inhibited. In the culture assay, E. multilocularis protoscoleces were treated with atovaquone (ATV), rotenone, praziquantel, artemisinin, and pyrvinium pamoate at a final concentration of 50 µM in different culture media. The viability of protoscoleces was compared under aerobic and anaerobic conditions via culture experiments. The survival days of E. multilocularis protoscoleces were evaluated in the drug-treated group compared with those in the non-treated group. The results of these culture assays revealed that praziquantel and artemisinin did not eliminate the protoscoleces under both aerobic and anaerobic conditions. However, a stronger elimination ability was observed with the co-administration of praziquantel or artemisinin with ATV than with ATV alone under aerobic conditions. Pyrvinium pamoate completely killed protoscoleces at 5 and 7 days under aerobic and anaerobic conditions, respectively. Pyrvinium pamoate behaved identically to rotenone, the complex I inhibitor, in the culture treatment assay. The data serve as a reference for the development of novel anti-echinococcal drugs.

Characterization of microRNAs expressed in the cystic legion of the liver of Mus musculus perorally infected with Echinococcus multilocularis Nemuro strain.

Alveolar echinococcosis (AE) is a zoonosis caused by the metacestode of Echinococcus multilocularis. The published genome of E. multilocularis showed that approximately 86% of its genome is non-coding. Micro RNAs (miRNAs) are small non-coding regulatory RNAs, and recent studies on parasitic helminths expect miRNAs as a promising target for drug development and diagnostic markers. Prior to this study, only a few studies reported the E. multilocularis miRNA profiles in the intermediate host. The primary objective of this study was to characterize miRNA profiles via small RNA-seq in E. multilocularis Nemuro strain, a laboratory strain of Asian genotype, using mice perorally infected with the parasite eggs. The data were then compared with two previously published small RNA-seq data. We identified 44 mature miRNAs as E. multilocularis origin out of the 68 mature miRNA sequences registered in the miRNA database miRbase. The highest quantities of miRNAs detected were miR-10-5p, followed by bantam-3p, let-7-5p, miR-61-3p, and miR-71-5p. The top two most abundant miRNAs (miR-10-5p and bantam-3p) accounted for approximately 80.9% of the total parasite miRNAs. The highly expressed miRNA repertoire is mostly comparable to that obtained from the previous experiment using secondary echinococcosis created by an intraperitoneal administration of metacestodes. A detailed characterization and functional annotations of these shared miRNAs will lead to a better understanding of parasitic dynamics, which could provide a basis for the development of novel diagnostic and treatment methods for AE.

Adult worm exclusion and histological data of dogs repeatedly infected with the cestode Echinococcus multilocularis.

The data presented in this article are related to a previously published research article titled "The timing of worm exclusion in dogs repeatedly infected with the cestode Echinococcus multilocularis" (2016) [1]. This data describe a comparison of worm exclusion in the early stage of infection (1 day and 6 days post-infection) between dogs infected for the first time (control group) and dogs repeatedly infected with the parasite 4 times (repeated infection groups). We observed that 6 days post reinfection, the number of adult worms in repeated-infection groups decreased by 88.7% compared with the control group. Histological analysis comparison of the small intestinal mucosa from healthy, first infected, and repeatedly infected dogs are also reported. We observed no clear pathological abnormality, except the shortening of microvillus in reinfected dogs. However, eosinophil accumulation and eosinophilic ulcers were observed in some reinfected dogs. This data could be useful as preliminary data to develop a final host vaccine for this parasite.

Mitochondrial complex III in larval stage of Echinococcus multilocularis as a potential chemotherapeutic target and in vivo efficacy of atovaquone against primary hydatid cysts.

Echinococcus multilocularis employs aerobic and anaerobic respiration pathways for its survival in the specialized environment of the host. Under anaerobic conditions, fumarate respiration has been identified as a promising target for drug development against E. multilocularis larvae, although the relevance of oxidative phosphorylation in its survival remains unclear. Here, we focused on the inhibition of mitochondrial cytochrome bc1 complex (complex III) and evaluated aerobic respiratory activity using mitochondrial fractions from E. multilocularis protoscoleces. An enzymatic assay revealed that the mitochondrial fractions possessed NADH-cytochrome c reductase (mitochondrial complexes I and III) and succinate-cytochrome c reductase (mitochondrial complexes II and III) activities in the aerobic pathway. Enzymatic analysis showed that atovaquone, a commercially available anti-malarial drug, inhibited mitochondrial complex III at 1.5 nM (IC50). In addition, culture experiments revealed the ability of atovaquone to kill protoscoleces under aerobic conditions, but not under anaerobic conditions, indicating that protoscoleces altered their respiration system to oxidative phosphorylation or fumarate respiration depending on the oxygen supply. Furthermore, combined administration of atovaquone with atpenin A5, a quinone binding site inhibitor of complex II, completely killed protoscoleces in the culture. Thus, inhibition of both complex II and complex III was essential for strong antiparasitic effect on E. multilocularis. Additionally, we demonstrated that oral administration of atovaquone significantly reduced primary alveolar hydatid cyst development in the mouse liver, compared with the untreated control, indicating that complex III is a promising target for development of anti-echinococcal drug.

First report of Sarcocystis pilosa sporocysts in feces from red fox, Vulpes vulpes schrencki, in Hokkaido, Japan.

Sarcocysts of various Sarcocystis spp. are highly prevalent in wild sika deer, Cervus nippon yesoensis, in Hokkaido, Japan, and four species have been identified based on morphological and molecular characteristics: S. ovalis, S. pilosa, S. tarandi-like, and S. truncata-like. The definitive hosts of S. ovalis are corvids, but the hosts of the other species have not yet been identified. Aiming to determine the definitive hosts of these species, we collected 65 red fox (Vulpes vulpes schrencki) fecal samples in eastern Hokkaido and examined them for fecal sporocysts using a modified sucrose flotation method. One fecal sample contained typical Sarcocystis sporocysts, which were identified as S. pilosa based on 18S ribosomal RNA and cytochrome c oxidase subunit I gene sequences. This is the first identification of S. pilosa sporocysts in the wild. These findings indicate that red foxes serve as a definitive host of S. pilosa, and that red foxes constitute a source of S. pilosa infection for deer in Hokkaido.

Molecular characterization of three Sarcocystis spp. from wild sika deer (Cervus nippon yesoensis) in Hokkaido, Japan.

Diaphragm samples from 65 hunted sika deer (Cervus nippon yesoensis) from Hokkaido, Japan were examined for the presence of sarcocysts based on histological sections. Morphologically, the detected sarcocysts grouped into three types: (Type 1) 108.0-305.0 µm in width, thick-walled (4.3-7.0 µm) with tombstone-like protrusions; (Type 2) 25.0-69.5 µm in width, thick-walled (3.8-8.0 µm) with finger-like protrusions; and (Type 3) 22.5-55.0 µm in width, thin-walled (under 1 µm) with no visible protrusions under light microscopy. All samples contained at least one sarcocyst type, and multiparasitism was apparent in 58 samples. Morphologically, Type 1 sarcocysts were found in 19 (29.2%) samples, Type 2 in 62 (95.4%) samples, and Type 3 in 60 (92.3%) samples. The sarcocysts were collected using laser microdissection, the DNA extracted from them was PCR-amplified, and their 18S ribosomal RNA and cytochrome c oxidase subunit 1 genes were sequenced. Phylogenetic analysis showed that, for both genes, each morphological sarcocyst type (Types 1, 2, and 3) aligned most closely with S. silva/S. truncata, S. tarandi/S. elongata, and S. pilosa, respectively. Based on the sequence identities between taxa and the molecular information for sarcocysts in C. nippon centralis, the sarcocyst types were presumed to be S. truncata-like (Type 1), S. tarandi-like (Type 2), and S. pilosa (Type 3). The phylogenetic analyses based on the present comprehensive molecular characterization of three Sarcocystis spp. from C. nippon yesoensis in Hokkaido suggest that canids (e.g., wild foxes) may be the definitive hosts for S. pilosa, and felids (or unknown species) the definitive hosts for the other two species.

High probability of pet dogs encountering the sylvatic cycle of Echinococcus multilocularis in a rural area in Hokkaido, Japan.

Surveillance of Echinococcus multilocularis in 98 pet dogs kept in a rural area of Hokkaido, Japan, from March 2018 to March 2019 suggested infection in seven dogs (7.1%) by E. multilocularis-specific copro-DNA examination, and one of them excreted E. multilocularis eggs that were identified by sequence analyses. Among the infected dogs, three were not allowed to run free when outdoors. Based on detection of E. multilocularis eggs in fox feces collected from roadsides in the same area, dogs kept in rural areas may have a high probability of becoming infected after preying on infected voles along such roadsides, even in domesticated settings. Therefore, examination along with periodic deworming administration is considered necessary to prevent transmission from dogs to owners.

Molecular and functional characterization of glucose transporter genes of the fox tapeworm Echinococcus multilocularis.

Alveolar echinococcosis (AE) is a zoonotic parasitosis caused by larvae of the fox tapeworm, Echinococcus multilocularis. E. multilocularis is distributed widely in the Northern hemisphere, causing serious health problems in various animals and humans. E. multilocularis, like other cestodes, lacks a digestive tract and absorbs essential nutrients, including glucose, across the syncytial tegument on its external surface. Therefore, it is hypothesized that E. multilocularis uses glucose transporters on its surface similar to a closely-related species, Taenia solium. Based on this hypothesis, we cloned and characterized glucose transporter homologues from E. multilocularis. As a result, we obtained full-length sequences of 2 putative glucose transporter genes (EmGLUT1 and EmGLUT2) from E. multilocularis. In silico analysis predicted that these were classified in the solute carrier family 2 group. Functional expression analysis using Xenopus oocytes demonstrated clear uptake of 2-deoxy-D-glucose (2-DG) by EmGLUT1, but not by EmGLUT2 in this experimental system. EmGLUT1 was shown to have relatively high glucose transport activity. Further analyses using the Xenopus oocyte system revealed that 2-DG uptake of EmGLUT1 did not depend on the presence or concentration of Na+ nor H+, respectively. Immunoblot analyses using cultured metacestode, ex vivo protoscolex, and adult worm samples demonstrated that both EmGLUTs were stably expressed during each developmental stage of the parasite. Based on the above-mentioned findings, we conclude that EmGLUT1 is a simple facilitated glucose transporter and possibly plays an important role in glucose uptake by E. multilocularis throughout its life cycle.

Analysis for genetic loci controlling protoscolex development in the Echinococcus multilocularis infection using congenic mice.

The resistance/susceptibility to Echinococcus multilocularis infection in mice is genetically controlled. However, genetic factors responsible for these differences remain unknown. Our previous study in genetic linkage analysis has revealed that there is a significant quantitative trait locus (QTL) for the establishment of cyst (Emcys1), and a highly significant QTL for the development of protoscolex of E. multilocularis larvae (Empsc1), on mouse chromosomes 6 and 1, respectively. The current study aimed to confirm these QTLs and narrow down the critical genetic region that controls resistance/susceptibility to E. multilocularis infection by establishing congenic and subcongenic lines from C57BL/6 (B6) and DBA/2 (D2) mice. For protoscolex development phenotype, two congenic lines, B6.D2-Empsc1 and D2.B6-Empsc1 were developed, where responsible QTL, Empsc1 was introgressed from D2 into B6 background and vice versa. For cyst establishment phenotype, two congenic lines, B6.D2-Emcys1 and D2.B6-Emcys1 were developed, where responsible QTL, Emcys1 was introgressed from D2 into B6 background and vice versa. Because there was no significant difference in cyst establishment between B6.D2-Emcys1 and D2.B6-Emcys1 mice after challenge with E. multilocularis, it is suggested that the Emcys1 does not solely control the cyst establishment in mouse liver. However, infection experiments with B6.D2-Empsc1 and D2.B6-Empsc1 mice showed a significant difference in protoscolex development in the cyst. It confirms that the Empsc1 controls phenotype of the protoscolex development in the cyst. Subsequently, two subcongenic lines, B6.D2-Empsc1.1 and B6.D2-Empsc1.2 from B6.D2-Emcys1 and one subcongenic line, D2.B6-Empsc1.1 from D2.B6-Empsc1 were developed to narrow down the critical region responsible for protoscolex development. From the results of infection experiments with E. multilocularis in these subcongenic mice, it is concluded that a gene responsible for protoscolex development is located between D1Mit290 (68.1 cM) and D1Mit511 (97.3 cM).

Gene expression profiles of the small intestinal mucosa of dogs repeatedly infected with the cestode Echinococcus multilocularis.

The data set presented in this article is related to a previous research article entitled " The timing of worm exclusion in dogs repeatedly infected with the cestode Echinococcus multilocularis" (Kouguchi et al., 2016) [1]. This article describes the genes >2-fold up- or down-regulated in the first- and repeated-infection groups compared to the healthy controls group. The gene expression profiles were generated using the Agilent-021193 Canine (V2) Gene Expression Microarray (GPL15379). The raw and normalized microarray data have been deposited with the Gene Expression Omnibus (GEO) database under accession number GSE105098.

Echinococcus multilocularis Surveillance Using Copro-DNA and Egg Examination of Shelter Dogs from an Endemic Area in Hokkaido, Japan.

Surveillance of Echinococcus multilocularis infection among 156 shelter dogs was conducted in an urban area (Sapporo city) in Hokkaido, where the parasite is endemic in Japan using copro-DNA and fecal egg examination from September 2013 to April 2017. Echinococcus infection was detected in three dogs (1.9%), including one dog that excreted eggs. The results suggested that free-roaming or stray dogs in urban area may be infected by capturing wild voles containing parasitic cysts and could be a source of human infection. Dog-to-human transmission is a significant concern, and the risk of such transmission is present even in urban areas in Hokkaido. We recommend deworming within 1 month (e.g., before egg excretion) of capture for free-roaming or stray dogs in Echinococcus-endemic area to prevent potential human infection.

Development of LAMP assays for the molecular detection of taeniid infection in canine in Tibetan rural area.

For field-identification of taeniid cestodes in canine animals in Tibetan area, loop-mediated isothermal amplification (LAMP) assays for Echinococcus multilocularis, E. shiquicus, Taenia hydatigena, T. multiceps, T. pisiformis and T. crassiceps were developed and evaluated along with the reported assay for E. granulosus. The LAMP assays showed specific reaction with their corresponding target species DNA with the detection limit of 1 to 10 pg. Moreover, the assays for E. granulosus, E. multilocularis, T. hydatigena and T. multiceps could detect DNA extracted from 3 or more eggs of their corresponding target species. Then, the LAMP assays were applied on samples containing 3 to 35 taeniid eggs obtained from 61 field-collected canine feces in Qinghai, and the result was compared with a reported multiplex PCR and sequence analysis. The LAMP assays and the PCR detected single species DNA of E. granulosus, E. shiquicus, T. hydatigena and T. multiceps in 5, 2, 44 and 2 samples, respectively. In the rest 8 samples, DNA of both E. granulosus and T. hydatigena were detected by the PCR but the LAMP assays detected those DNAs in 2 samples and only T. hydatigena DNA in 6 samples. It was assumed that less than 3 E. granulosus eggs were mixed in the samples although the samples contained 21 to 27 eggs in total. In conclusion, the LAMP assays were less sensitive than the multiplex PCR, but would have adequate sensitivity for field use in Tibetan area.