Genetic diversity and haplotypes of Echinococcus granulosus isolated from cattle and buffaloes and first report of E. ortleppi (G5) in buffaloes in Pakistan based on mitochondrial cytochrome c oxidase subunit-1 gene (mt-CO1) markers.

Cystic echinococcosis (CE) is a parasitic disease that is caused by larval stage of Echinococcus granulosus tapeworm, one of the most important and neglected zoonotic disease. Although the echinococcosis is endemic in the neighboring countries, information regarding circulating genotypes of E. granulosus sensu lato is scarce in Pakistan. Therefore, the main purpose of this report was to contribute in molecular epidemiology and to find genetic variation and haplotypes of E. granulosus s.l. in cattle and buffalo isolates. To identify species circulating in country, parasite samples were collected from different slaughterhouses and butcher shops of two major cities, Rawalpindi and Peshawar located in Punjab and Khyber Pakhtunkhwa (KP) provinces, Pakistan, respectively. A total of 100 CE cyst samples were investigated from buffalo (n = 61), and cattle (n = 39) hosts. After genomic DNA extraction from individual cyst materials, mt-CO1 (875 bp) gene was amplified by PCR. After that, PCR products were electrophoresed on the agarose gel then purified and sequenced using forward primer. The sequences were trimmed (779 bp), aligned and matched with NCBI published sequences. E. granulosus s.s. (G1, G3) (71.4%; n = 20/28) was confirmed as the dominant species in buffalo and cattle. E. ortleppi (G5) (28.6%; n = 8/28) was recorded for the first time in both buffalo and cattle isolates from Rawalpindi. E. granulosus s.l. haplotype network showed single predominant haplotype, which comprised 40% of population. Tajima's D and Fu's Fs were negative and significant for E. ortleppi (G5), suggesting population expansion in Pakistan. Therefore, more studies using isolates of E. granulosus s.l. from various locations and intermediate hosts across Pakistan will add new data on molecular epidemiology and genotyping for effective control strategies of CE in Pakistan.

Sensitive detection of specific cell-free DNA in serum samples from sheep with cystic echinococcosis.

BACKGROUND: Developing more sensitive methods for the diagnosis of echinococcosis is essential. In this study PCR assay for sensitive detection of specific cell-free DNA (cfDNA) of Echinococcus granulosus sensu lato in the sera of the sheep naturally infected with echinococcosis was investigated. METHODS: To extract cfDNA from 35 infected sheep, the modified phenol-chloroform method was used for two different volumes (0.5 and 2 ml) of serum samples. From each extracted sample, two DNA volumes (5 and 10 µl) were amplified using both standard PCR and semi-nested PCR targeting NADH dehydrogenase subunit I. RESULTS: Standard and semi-nested PCR on 0.5 ml of serum samples detected Echinococcus DNA in 8 and 12 out of 35 sheep, respectively; however, using 2 ml of serum samples, they detected 24 and 27 samples. By increasing the volume of template DNA, the PCRs could detect 29 and 33 out of 35 samples. The results were confirmed by sequencing of randomly selected PCR amplicons and comparing them with GenBank databases. CONCLUSIONS: Larger volumes of serum for DNA extraction, greater volumes of DNA template for PCR, and employing a semi-nested PCR protocol, increased the sensitivity of PCR to 95%. This approach can also be applied to the diagnosis of echinococcosis in humans.

Interplays of liver fibrosis-associated microRNAs: Molecular mechanisms and implications in diagnosis and therapy.

microRNAs (miRNAs) are a class of non-coding functional small RNA composed of 21-23 nucleotides, having multiple associations with liver fibrosis. Fibrosis-associated miRNAs are roughly classified into pro-fibrosis or anti-fibrosis types. The former is capable of activating hepatic stellate cells (HSCs) by modulating pro-fibrotic signaling pathways, mainly including TGF-ß/SMAD, WNT/ß-catenin, and Hedgehog; the latter is responsible for maintenance of the quiescent phenotype of normal HSCs, phenotypic reversion of activated HSCs (aHSCs), inhibition of HSCs proliferation and suppression of the extracellular matrix-associated gene expression. Moreover, several miRNAs are involved in regulation of liver fibrosis via alternative mechanisms, such as interacting between hepatocytes and other liver cells via exosomes and increasing autophagy of aHSCs. Thus, understanding the role of these miRNAs may provide new avenues for the development of novel interventions against hepatic fibrosis.

Design of ion channel blocking, toxin-like Kunitz inhibitor peptides from the tapeworm, Echinococcus granulosus, with potential anti-cancer activity.

Over-expression of K+ channels has been reported in human cancers and is associated with the poor prognosis of several malignancies. EAG1, a particular potassium ion channel, is widely expressed in the brain but poorly expressed in other normal tissues. Kunitz proteins are dominant in metazoan including the dog tapeworm, Echinococcus granulosus. Using computational analyses on one A-type potassium channel, EAG1, and in vitro cellular methods, including major cancer cell biomarkers expression, immunocytochemistry and whole-cell patch clamp, we demonstrated the anti-tumor activity of three synthetic small peptides derived from E. granulosus Kunitz4 protease inhibitors. Experiments showed induced significant apoptosis and inhibition of proliferation in both cancer cell lines via disruption in cell-cycle transition from the G0/G1 to S phase. Western blotting showed that the levels of cell cycle-related proteins including P27 and P53 were altered upon kunitz4-a and kunitz4-c treatment. Patch clamp analysis demonstrated a significant increase in spontaneous firing frequency in Purkinje neurons, and exposure to kunitz4-c was associated with an increase in the number of rebound action potentials after hyperpolarized current. This noteworthy component in nature could act as an ion channel blocker and is a potential candidate for cancer chemotherapy based on potassium channel blockage.

MicroRNA-Transcription factor regulatory networks in the early strobilar development of Echinococcus granulosus protoscoleces.

BACKGROUND: Echinococcus granulosus sensu lato has a complex developmental biology with a variety of factors relating to both intermediate and final hosts. To achieve maximum parasite adaptability, the development of the cestode is dependent on essential changes in transcript regulation. Transcription factors (TFs) and miRNAs are known as master regulators that affect the expression of downstream genes through a wide range of metabolic and signaling pathways. In this study, we aimed to develop a regulatory miRNA-Transcription factor (miRNA-TF) network across early developmental stages of E. granulosus protoscoleces by performing in silico analysis, and to experimentally validate TFs expression in protoscoleces obtained from in vitro culture, and from in vivo experiments. RESULTS: We obtained list of 394 unique E. granulosus TFs and matched them with 818 differentially expressed genes which identified 41 predicted TFs with differential expression. These TFs were used to predict the potential targets of 31 differentially expressed miRNAs. As a result, eight miRNAs and eight TFs were found, and the predicted network was constructed using Cytoscape. At least four miRNAs (egr-miR-124a, egr-miR-124b-3p, egr-miR-745-3p, and egr-miR-87-3p) and their corresponding differentially expressed TFs (Zinc finger protein 45, Early growth response protein 3, Ecdysone induced protein 78c and ETS transcription factor elf 2) were highlighted in this investigation. The expression of predicted differentially expressed TFs obtained from in vitro and in vivo experiments, were experimentally validated by quantitative polymerase chain reaction. This confirmed findings of RNA-seq data. CONCLUSION: miRNA-TF networks presented in this study control some of the most important metabolic and signaling pathways in the development and life cycle of E. granulosus, providing a potential approach for disrupting the early hours of dog infection and preventing the development of the helminth in the final host.

Trichinella infections in animals and humans of Iran and Turkey.

Trichinellosis is considered as a cosmopolitan zoonosis caused by different species of the small nematodes of the genus Trichinella. The present study aimed to provide a broad review for exploring Trichinella sp. infection in humans and animals of Iran and Turkey. Additionally, we aimed to explore bases for trichinellosis prevention and control. Two reports of human trichinellosis following the consumption of meat of wild boar are available in the northern Iran. A large outbreak of trichinellosis and some other sporadic cases are reported mainly as a result of eating wild boar or pork meat from Turkey, where T. britovi is present. Field studies show that Trichinella sp. infections occur in wild carnivores of Iran, particularly the golden jackal (Canis aureus) as the most frequently infected species. T. britovi has been reported to be present elsewhere in Iran in wild mammals, where wild boar is the main source of Trichinella sp. infection. In Turkey, Trichinella spp. has been reported from animals including both domesticated and wild pigs and gray wolf (Canis lupus). However, current data on the distribution of Trichinella taxa are fragmentary in the Anatolian region.

Anti-echinococcal effects of sumac, Rhus coriaria, in a murine model of cystic echinococcosis: Parasitological and molecular evaluation.

Sumac has been traditionally used by people as a medicinal plant for the treatment of different disorders. Cystic echinococcosis (CE) is one of the major zoonotic diseases of human with a worldwide distribution. Long term albendazole therapy is usually associated with side effects including impaired liver function and leukopenia. The present study investigated the efficacy of the methanolic extract of Sumac, Rhus coriaria, on the secondary hydatid cyst development in mice and evaluated sumac effects on the expression of a profile of genes with a potential role in parasite development. Thirty-six mice were intraperitoneally inoculated with of with 3000 protoscoleces and six months after induction of infection were divided into three groups that received either oral sumac extract, albendazole or distilled water. The mice were necropsied 45 days later and the volume and weight of cysts were measured. The expression level of five target genes were analyzed using RT-qPCR. The volume and weight of the cysts were significantly lower in the sumac group compared to the controls. Decreased expressions were found in four out of the five genes following sumac administration. While significantly lower expressions in the sumac group were found for the cdk6, b-raf, fgfr and ras genes, no significant difference was found in cdk2 expression as compared with the control groups. Findings of the present study indicate high efficacy of sumac on the size and volume of secondary hydatid cysts in a murine CE model. Further studies are required to explore the most active and effective ingredients of this natural product.

Molecular identification and phylogenetic analysis of free-living amoeba in the water resources of Arak, Iran.

The aim of the present study was to detect free-living amoeba (FLA) in the water resources of Arak, Iran using molecular tools. A total of 154 samples were collected from different water supplies. Molecular analyses, sequencing, and phylogenetic study were conducted to confirm the species and genotypes of FLA. Fisher exact test was used to determine the significance. Of 154 water samples, 19 (12.3%) samples were tested positive for FLA. Three genotypes of Acanthamoeba including T4, subtype D, and T5 were identified among the isolates. The pathogenicity assay showed that the isolate of Acanthamoeba in drinking water was highly pathogenic. Three species of Naegleria, including N. australiensis, N. pagei, and N. gruberi were found among the samples. Six isolates of Vermamoeba were identified as V. vermiformis. Meanwhile, three other species including Vannella sp., Vahlkampfia avara, and Stenamoeba polymorpha were also recovered from the water samples. Statistical analysis showed a significant difference between the various water resources contaminated with FLA. This is the first study to reveal the presence of S. polymorpha in water sources in Iran. According to the findings of the present study, health officials should be beware of potential public health impacts of FLA in water resources.

Echinococcosis in immunocompromised patients: A systematic review.

BACKGROUND: Human echinococcoses are the infection caused by the larval stages of different species of the genus Echinococcus, mostly E. granulosus and E. multilocularis. There is no aggregated information on the nature and characteristics echinococcosis in patients with immunodeficiency. This study presents a systematic review of the current literature published on the status of echinococcosis in immunocompromised individuals. METHODS: An electronic search of related articles in four major databases (PubMed, Scopus, Web of Science and Google Scholar) was performed up to November 2021. All related studies meeting the inclusion criteria were assessed for qualitative analysis. Data available on different characteristics of the diseases were extracted. The data were subsequently categorized into two subgroups: Cystic Echinococcosis (CE) and Alveolar Echinococcosis (AE). RESULTS: Twenty-eight articles related to the existence of echinococcosis in immunocompromised hosts were included. HIV/AIDS was found as the most frequent condition in immunocompromised CE patients. Most of the CE cases with immunodeficiency were female (66.4%). The dominant stages of the cysts were CE2 and CE3. Surgery was performed for 76.2% of the patients. A high mortality rate of 23.8% was recorded in CE patients. Malignancies was the dominant condition in AE patients. CONCLUSION: Findings of the present study can potentially improve our understanding of the impact of immunodeficiency syndromes on echinococcoses and contribute to an improved diagnosis, treatment and quality of care in immunocompromised patients suffering from cystic and alveolar echinococcosis.

Efficient delivery of Echinococcus multilocularis miRNAs using chitosan nanoparticles.

Alveolar echinococcosis caused by Echinococcus multilocularis is an important zoonotic disease, a great threat to human health due to limited interventions. microRNAs are a type of small non-coding RNA that plays a key role in many diseases and is considered as a potential therapeutic target for control of parasitic diseases. However, naked miRNAs are difficult to enter into cells and are easily degraded in both external and internal environments. Chitosan (CS) has recently been used as a promising vehicle for delivery of nucleic acids. Therefore, we prepared miRNA-bearing CS nanoparticles and investigated the physicochemical properties as well as the delivery efficiency. We found that CS nanoparticles was relatively stable, offered miRNA strong protection from degradation and had low cytotoxicity with no significant effects on cell proliferation and apoptosis. CS nanoparticles were shown to be easily absorbed by cells and have remarkable liver tropism. Furthermore, CS nanoparticles were used to efficiently deliver E. multilocularis miR-4989 in vitro and in vivo and caused a significant reduction in the expression of UBE2N in the liver, a potential target of emu-miR-4989, at both mRNA and protein levels. Our data demonstrate that CS nanoparticles can act as a vehicle for efficient liver-targeted delivery of miRNAs and for development of miRNA-based therapeutics against E. multilocularis infection.

Dynamic modeling of female neutering interventions for free-roaming dog population management in an urban setting of southeastern Iran.

Understanding dynamics of free-roaming dog (FRD) population is critical for planning and implementation of dog population management programs. FRD population size estimation as well as dynamic modeling of dog population under different female dog neutering interventions were investigated in order to determine the most appropriate animal birth control approach. We performed population size estimate of dogs using sight-resight surveys by photography in a randomly selected 25 blocks of the city and all the suburbs of greater Kerman area. Main demographic features were characterized and the dog density distribution was mapped. A dynamic model was developed to predict free-roaming dog population variations after 5 and 10 years. Different scenarios based on 10, 30, 50, 60 and 70% female dog sterilization were considered to predict the effects of animal birth control measures. Free roaming dog population was estimated at 6781 dogs (65.3% males) in Kerman and suburbs with several major population hotspots. Analysis of the dog locations within the city showed that the largest proportion of the dogs were observed in the vacant lots (46.2%). Modeling predictions indicated that, in the absence of management, the free-roaming dog population could increase from a baseline of 6781 to 13,665 dogs (2.02 fold increase) in 5 years and to 19,376 dogs in 10 years (2.86 fold increase). Using a population dynamics model, we simulated five neutering coverages to explore the impact of female neutering on free-roaming dog population size. The 5-year projections of the model have shown that 50% annual female dog sterilization significantly reduced free-roaming dog population by 0.44 comparing to the baseline population. Findings of the present study improve our knowledge on the nature and extent of dog population dynamics in Iran. Effective population control and selection of the most appropriate neutering interventions require a comprehensive knowledge of the characteristics and dynamics of FRD population.

A Retrospective Cohort Study on Human Cystic Echinococcosis in Khyber Pakhtunkhwa Province (Pakistan) Based on 16 Years of Hospital Discharge Records.

Human cystic echinococcosis (CE) is a worldwide-distributed parasitic zoonotic disease, which represents a threat for both human and animals. The current study aimed at estimating the prevalence of human CE in Khyber Pakhtunkhwa (KPK) province of Pakistan. Clinical records from four major hospitals in this region were reviewed for CE human cases during the period of 2006-2021. Out of 251 (0.00071%) CE patients identified during the considered period, 142 (56.6%) were females, and 109 (43.4%) were males. The highest number of CE cases was recorded in the 21-30 (27.9%) age group, followed by 31-40 (23.1%) and 41-50 (16.3%). Most of the CE patients in KPK province were members of the Afghani ethnic group (17.1%); secondarily, they were Pakistani (6.4%), while for 76.5% ethnicity data were not available. The liver (41%) and the lungs (4.8%) were the most infected organs identified among CE patients in KPK province. The present study identified CE as a significant public health problem in KPK province, and the current findings demonstrated a constant endemicity of CE during the last 15 years. Further filed studies on the active search of CE carriers by means of ultrasound population-based surveys are needed to fill knowledge gaps on clinical and molecular epidemiology of human CE in Pakistan.

Genome-wide transcriptome analysis of the early developmental stages of Echinococcus granulosus protoscoleces reveals extensive alternative splicing events in the spliceosome pathway.

BACKGROUND: The complex life cycle of Echinococcus granulosus involves numerous environmental conditions within different intermediate and definitive hosts. This requires adaptation at different levels of transcript regulation. Alternative splicing (AS) and the related cellular functions as one of the major fields of post-genomics has been poorly studied in tapeworms. In the present study, we investigated AS events and their potential biological effects in E. granulosus. METHODS: Whole transcriptome sequencing data of four groups of protoscoleces were prepared for RNA-seq library construction. Fresh protoscoleces were either used as non-induced controls (NT group) or incubated for 15 min with pepsin (PEP group) and cultivated in a biphasic medium for 12 and 24 h (12 and 24 h groups). The frequency and different types of AS events were identified using rMATS software. Functional annotations and gene ontology of differential AS (DAS) genes were performed using Blast2GO software. AS events were experimentally validated by PCR on the protoscolex cDNAs using specific primers for each gene. RESULTS: At least one AS event was found in 38.1% of the genes (3904 out of 10,245) in the protoscoleces during early strobilar development. The genes were associated primarily with cellular and metabolic processes and binding and catalytic activity. KEGG pathway analysis of DAS events revealed a number of genes belonging to different components of the spliceosome complex. These genes tended to belong to common SR proteins, U1-related factors, U2-related factors, complex A-specific factors and other splicing-related proteins. CONCLUSIONS: The high number of AS events in the transcriptome regulatory mechanisms indicates the essential rapid molecular processes required by the parasite for adaptation in different environments.

MicroRNA profile of the strobilated worms of Echinococcus granulosus derived from in vivo and in vitro systems by using high-throughput approach.

MicroRNAs are critical gene regulators at the post-transcriptional level and play essential roles in numerous developmental processes in metazoan parasites including the causative agent of cystic echinococcosis, Echinococcus granulosus. The molecular basis of different patterns of E. granulosus development in the canine definitive host and in in vitro culture systems is poorly understood. In the present study, miRNA transcriptomes of the strobilated worms derived from experimental infection in the definitive host were compared with those from diphasic culture system after 60-day protoscoleces cultivation. Total RNA was extracted from in vivo- and in vitro-derived strobilated worms. Small RNA libraries were constructed, and deep sequencing was performed. Subsequently, differential miRNA expressions and target predictions were obtained, and pathway analysis was performed by gene ontology and KEGG. Seven miRNAs were differentially expressed between the in vivo- and in vitro-derived worms. In addition, we reported 13 novel miRNA candidates and 42 conserved miRNAs. Four out of five top miRNAs with the highest read counts were shared between the in vivo and in vitro-derived worms, i.e., egr-miR-10a-5p, egr-let-7-5p, egr-bantam-3p, and egr-miR-71-5p. Target prediction of the differential miRNAs between the two systems showed significant differences in the membrane-enclosed lumen, membrane part, and an intrinsic component of the membrane. Findings of KEGG analysis indicated that differentially expressed miRNAs were involved in hippo, MAPK, and WNT signaling pathways. The study demonstrated a significant difference in miRNA transcriptomes and related signaling pathways between the two systems, suggesting the importance of host-parasite interplay in the fate of protoscoleces development in in vivo and in vitro systems.

Molecular cloning and functional characterization of a thioredoxin peroxidase gene in Echinococcus multilocularis.

Thioredoxin peroxidase (TPx) plays an important role in protecting parasites against oxidative damage. However, studies on the role of TPxs in Echinococcus multilocularis are limited. In this study, one tpx gene of E. multilocularis, named as emtpx-1, was identified. EmTPx-1 shares two positionally conserved cysteine residues (Cys48 and Cys169) with orthologs from other platyhelminths. EmTPx-1 is highly expressed in the germinal layer and present in exosome-like vesicles secreted by E. multilocularis metacestodes. EmTPx-1 displays peroxidase activity, which removes hydrogen peroxide in the presence of dithiothreitol. Furthermore, EmTPx-1 could protect DNA from oxidative damages, and EmTPx-1-expressing E. coli cells had an enhanced resistance to oxidative stress. In addition, EmTPx-1 enhanced the expression of arg1, ym1, and il-10, but suppressed inos, tnf-α, and il-1ß expression in LPS-stimulated macrophages. Our data suggest a critical role for EmTPx-1 in oxidative stresses and M2 macrophage polarization.

Echinococcoses in Iran, Turkey, and Pakistan: Old Diseases in the New Millennium.

Echinococcosis is considered a cosmopolitan zoonosis caused by different species of small taeniid tapeworms of the genus Echinococcus and is regarded as a neglected zoonosis. Cystic and alveolar echinococcoses are endemic diseases of Tibetan, Pamir, and Iranian plateaus. All of the countries within the Iranian plateau are affected by echinococcosis. Pakistan, Turkey, and Iran are the three most populous countries of the region, in which echinococcosis is highly endemic. The three neighboring countries share strong cultural and socioeconomic ties. The present study aimed to provide a broad review of the status of cystic and alveolar echinococcosis, summarizing the current knowledge about geographical distribution, molecular epidemiology, and transmission dynamics of Echinococcus granulosus sensu lato and Echinococcus multilocularis in this region. Additionally, we aimed to understand disease burden and risk factors as basic requirements for establishing a surveillance system and planning prevention and control programs. A considerable body of information is available on different aspects of echinococcosis in this region; however, several information and research gaps need to be filled before planning control programs. None of the countries in the region have an elaborate echinococcosis control program. Effective control programs require multi/intersectoral coordination within a One Health approach with a long-term political and administrative commitment and enhanced international collaboration among the three countries.

Comparative Analysis of miRNA Expressions in Different Developmental Stages of Echinococcus granulosus in Mono-Phasic and Di-Phasic Culture Systems.

BACKGROUND: The dog tapeworm, Echinococcus granulosus, is a zoonotic parasite affecting humans and livestock across the globe. Basic research on the molecular biology and genetics of E. granulosus improves our understanding of the biology and potential drug targets in various developmental stages of E. granulosus in both definitive and intermediate hosts. There has been increasing interest in the identification of microRNAs in parasitic organisms. The purpose of the current study was to compare the activity of a selected profile of miRNAs in different developmental stages of E. granulosus. METHODS: Different developmental stages of the parasite were obtained from ex vivo as well as in vitro cultured E. granulosus. MicroRNAs were extracted from the ex vivo germinal layer and invaginated protoscoleces as well as the in vitro generated microcysts, evaginated protoscoleces, and strobilated worms. The expression of the selected miRNAs was evaluated by RT-qPCR for each stage. RESULTS: Four out of five miRNAs were present and active in different developmental stages of E. granulosus. A significant over-expression of miR-61 was observed in the germinal layer and during the protoscolex transformation into the microcysts, however, miR-10 was more expressed in the mature strobilated forms than in the other stages. Let-7 and miR-3489 showed a high expression in the germinal layer. CONCLUSION: Differential expression of four miRNAs among different in vitro and ex vivo developmental stages of E. granulosus was documented in the present study. Further experimental investigations are required to elucidate the probable role of the miRNAs in bi-directional differentiation of protoscoleces either into the strobilated worm or to a secondary hydatid cyst and the potential of these miRNAs as drug targets.

Quantifying the load of Echinococcus granulosus eggs in experimental dog infection using probe-based copro-qPCR analysis.

Designing and implementing Cystic Echinococcosis control programs require quantitative information about the worm load and the intensity of infection in dog populations in endemic areas. So far no "probe-based" molecular quantification tool has been available for E. granulosus. This study was conducted in order to develop and evaluate a qPCR technique for measuring worm load of E. granulosus in the final host. A species-specific TaqMan probe was designed based on the available sequences in GenBank. The study was conducted in two stages. First, stool samples from an experimentally infected dog were collected in days 7, 14, 21, 28, 35 and 49, and were analyzed by real-time qPCR assay. In the second stage, 600 mg negative stool specimens were manually spiked with 1, 5, 10, 20 and 40 eggs and the specimens were analyzed using real-time qPCR. According to the standard curve analysis, 93% efficiency and coefficients of correlation (Rsq) > 0.991 were documented. Quantitative PCR assays showed an increasing signal of infection during the 7-week course of infection. As revealed by the qPCR results from week 5 onward, signals indicative of egg excretion began and reached maximum on week 7. No qPCR signal from the samples containing 1, 10 and 20 eggs was recorded, however the samples containing 5 and 40 eggs produced signals proportional to the primary DNA. The study presents a molecular tool to quantify the burden of E. granulosus infection in dogs. This tool could be applied for measuring the burden of infection in the definitive hosts in surveillance and control programs.