Molecular characteristics of Echinococcus multilocularis FABP1 and its regulatory functions on murine macrophages.

Fatty acid binding proteins (FABPs) have emerged as attractive vaccination candidates for several platyhelminth species. To explore the physiological functions of Echinococcus multilocularis (E. multilocularis) FABP, the molecular characteristics of EmFABP1 were analyzed by online software, and the regulatory roles of rEmFABP1 protein in murine macrophages were further investigated. The emfabp1 gene encodes 133 amino acids with the characteristic ß-barrel shape of the cytoplasmic FABP family. Natural EmFABP1 protein is predominantly expressed in protoscoleces tegument and germinal layer cells and is also detected in cyst fluid and exosomes of E. multilocularis. rEmFABP1 protein demonstrated a notable suppression of phagocytic activity and nitric oxide production in murine macrophages. Additionally, the protein was observed to promote apoptosis and regulate cytokine expression in macrophages. These findings suggested that E. multilocularis FABP1 is critical in modifying macrophage physiological processes and that this protein may have immunomodulatory roles during infection.

Echinococcus granulosus Sensu Stricto and Echinococcus multilocularis in a Gray Wolf (Canis lupus) in Turkey: Further Evidence for Increased Risk of Alveolar Echinococcosis in Urban Areas.

OBJECTIVE: The aim of this study was to identify Echinococcus species by morphological and molecular means. METHODS: A dead gray wolf (Canis lupus) was found near Erzurum province and brought to the parasitology laboratory. Sedimentation and counting technique (SCT) and polymerase chain reaction (PCR) analysis were conducted. RESULTS: The SCT implications indicated that the wolf had a substantial worm burden (62,720 and 49,280 parasites) due to a co-infection of E. granulosus s.l. and E. multilocularis. Genus/species-specific PCR was used to analyze DNA extracted from adult worms and confirmed as E. granulosus s.s. and E. multilocularis, utilizing COI and 12S rRNA gene sequence analysis, respectively. CONCLUSION: This report presents the first co-detection of E. granulosus s.s. and E. multilocularis in a gray wolf found in an urban area in a highly endemic area for human echinococcosis in northeastern Turkey. The results emphasize that AE is not only a problem of rural areas, but also occurs in urban areas, which may pose a threat to public health. Therefore, surveillance in urban areas is crucial. The need to develop new control strategies for domestic and wildlife in the study area is also highlighted.

Study on the mechanism of miRNAs on liver injury in the condition of Protoscocephalus alveolarus transhepatic portal vein infection.

OBJECTIVE: To explore the role of miRNA in liver damage caused by Echinococcus multilocularis infection. METHODS: Six female C57BL mice were randomly divided into two groups, the control group and the infection group. Mice in the control group were injected with 100 µL PBS through the hepatic portal vein, and mice in the infection group were infected with E. multilocularis via the hepatic portal vein to establish a mouse model of infection. Small RNA sequencing was performed for detecting the expression of miRNAs in the liver of mice infected with 2000 E. multilocularis after 3 months of infection, screen out miRNAs related to liver damage, and verify by RT-PCR. RESULTS: Seventy-one differentially expressed miRNAs were found in the liver in comparison with control, and a total of 36 mouse miRNAs with |FC| >0.585 were screened out, respectively. In addition, Targetscan (V5.0) and miRanda (v3.3a) software were used to predict differential miRNAs target genes and functional enrichment of target genes. Functional annotation showed that "cytokine-cytokine interaction," "positive regulation of cytokine production," "inflammatory response," and "leukocyte activation" were enriched in the liver of E. multilocularis-infected mice. Moreover, the pathways "human cytomegalovirus infection," "cysteine and methionine metabolism," "Notch signaling pathway," and "ferroptosis" were involved in liver disease. Furthermore, four miRNAs (mmu-miR-30e-3p, mmu-miR-203-3p, mmu-miR-125b-5p, and mmu-miR-30c-2-3p) related to liver injury were screened and verified. CONCLUSION: This study revealed that the expression profiling of miRNAs in the livers was changed after E. multilocularis infection, and improved our understanding of the transcriptomic landscape of hepatic echinococcosis in mice.

[Effect of LAG3 deficiency on natural killer cell function and hepatic fibrosis in mice infected with Echinococcus multilocularis].

OBJECTIVE: To investigate the effect of LAG-3 deficiency (LAG3-/-) on natural killer (NK) cell function and hepatic fibrosis in mice infected with Echinococcus multilocularis. METHODS: C57BL/6 mice, each weighing (20 ± 2) g, were divided into the LAG3-/- and wild type (WT) groups, and each mouse in both groups was inoculated with 3 000 E. multilocularis protoscoleces via the hepatic portal vein. Mouse liver and spleen specimens were collected 12 weeks post-infection, sectioned and stained with sirius red, and the hepatic lesions and fibrosis were observed. Mouse hepatic and splenic lymphocytes were isolated, and flow cytometry was performed to detect the proportions of hepatic and splenic NK cells, the expression of CD44, CD25 and CD69 molecules on NK cell surface, and the secretion of interferon γ (IFN-γ), tumor necrosis factor α (TNF-α), interleukin (IL)-4, IL-10 and IL-17A. RESULTS: Sirius red staining showed widening of inflammatory cell bands and hyperplasia of fibrotic connective tissues around mouse hepatic lesions, as well as increased deposition of collagen fibers in the LAG3-/-group relative to the WT group. Flow cytometry revealed lower proportions of mouse hepatic (6.29% ± 1.06% vs. 11.91% ± 1.85%, P < 0.000 1) and splenic NK cells (4.44% ± 1.22% vs. 5.85% ± 1.10%, P > 0.05) in the LAG3-/- group than in the WT group, and the mean fluorescence intensity of CD44 was higher on the surface of mouse hepatic NK cells in the LAG3-/- group than in the WT group (t = -3.234, P < 0.01), while no significant differences were found in the mean fluorescence intensity of CD25 or CD69 on the surface of mouse hepaticNK cells between the LAG3-/- and WT groups (both P values > 0.05). There were significant differences between the LAG3-/- and WT groups in terms of the percentages of IFN-γ (t = -0.723, P > 0.05), TNF-α (t = -0.659, P > 0.05), IL-4 (t = -0.263, P > 0.05), IL-10 (t = -0.455, P > 0.05) or IL-17A secreted by mouse hepatic NK cells (t = 0.091, P > 0.05), and the percentage of IFN-γ secreted by mouse splenic NK cells was higher in the LAG3-/- group than in the WT group (58.40% ± 1.64% vs. 50.40% ± 4.13%; t = -4.042, P < 0.01); however, there were no significant differences between the two groups in terms of the proportions of TNF-α (t = -1.902, P > 0.05), IL-4 (t = -1.333, P > 0.05), IL-10 (t = -1.356, P > 0.05) or IL-17A secreted by mouse splenic NK cells (t = 0.529, P > 0.05). CONCLUSIONS: During the course of E. multilocularis infections, LAG3-/- promotes high-level secretion of IFN-γ by splenic NK cells, which may participate in the reversal the immune function of NK cells, resulting in aggravation of hepatic fibrosis.

The A2 haplotype of Echinococcus multilocularis is the predominant variant infecting humans and dogs in Yili Prefecture, Xinjiang.

Alveolar echinococcosis (AE), caused by Echinococcus multilocularis, is an important zoonotic disease. Yili Prefecture in Xinjiang is endemic for AE, however the molecular variability of E. multilocularis in this region is poorly understood. In this study, 127 samples were used for haplotypes analysis, including 79 tissues from humans, 43 liver tissues from small rodents, and 5 fecal samples from dogs. Genetic variability in E. multilocularis was studied using complete sequences of the mitochondrial (mt) genes of cytochrome b (cob), NADH dehydrogenase subunit 2 (nad2), and cytochrome c oxidase subunit 1 (cox1), using a total of 3558 bp per sample. The Asia haplotype 2 (A2) was the dominant haplotype, with 72.15% (57/79) prevalence in humans, 2.33% (1/43) in small rodents, and 80.00% (4/5) in dogs, followed by A5, the second most common haplotype, which infected 27.91% (12/43) small rodents. Haplotype network analysis showed that all haplotypes clustered together with the Asian group. Pairwise fixation index (FST) values showed lower level of genetic differentiation between different regions within the country. Compared with the sequences of E. multilocularis from North America and Europe, all concatenated sequences isolated from Yili Prefecture were highly differentiated and formed a single population. The A2 haplotype, analyzed using the cob, nad2, and cox1 genes of E. multilocularis, is the predominant variant in humans and dogs in Yili Prefecture.

Molecular analyses of exosome-derived miRNAs revealed reduced expression of miR-184-3p and decreased exosome concentration in patients with alveolar echinococcosis.

Both E. multilocularis and host-derived exosomes are involved in the pathogenic process of alveolar echinococcosis (AE). Exosomes secrete miRNAs that have regulatory roles in host-pathogen interactions in multiple ways. In the present study, we collected and purified supernatants of E. multilocularis cultures, as well as human plasma exosomes. High-throughput sequencing showed the identities of 45 exosomal miRNAs in E. multilocularis. The lengths of these miRNAs ranged from 19 to 25 nucleotides (nt), with the majority (n = 18) measuring 22 nt. Notably, emu-let-7-5p emerged as the most abundant among these miRNAs, with a detected count of 33,097 and also length of 22 nt. Nanoparticle tracking analysis (NTA) showed that the concentration of exosomes in the plasma of AE patients was lower compared to that in the healthy individuals. This result suggested that the concentration of plasma exosomes was able to distinguish AE patients from healthy individuals. Using qRT-PCR to assess the relative expression of 10 miRNAs of E. multilocularis, we showed that the expression of miR-184-3p was downregulated significantly in the exosomes of plasma from AE patients compared to that in the control group. In summary, this study indicates that AE induces a reduction in the concentration of human plasma exosomes, as well as downregulating miR-184-3p in infected individuals.

Assessing the role of individual foxes in environmental contamination with Echinococcus multilocularis through faecal samples.

Key parasite transmission parameters are difficult to obtain from elusive wild animals. For Echinococcus multilocularis, the causative agent of alveolar echinococcosis (AE), the red fox is responsible for most of the environmental contamination in Europe. The identification of individual spreaders of E. multilocularis environmental contamination is crucial to improving our understanding of the ecology of parasite transmission in areas of high endemicity and optimising the effectiveness of prevention and control measures in the field. Genetic faecal sampling appears to be a feasible method to gain information about the faecal deposition of individual animals. We conducted a 4 year faecal sampling study in a village that is highly endemic for E. multilocularis, to assess the feasibility of individual identification and sexing of foxes to describe individual infection patterns. Individual fox identification from faecal samples was performed by obtaining reliable genotypes from 14 microsatellites and one sex locus, coupled with the detection of E. multilocularis DNA, first using captive foxes and then by environmental sampling. From a collection of 386 fox stools collected between 2017 and 2020, tested for the presence of E. multilocularis DNA, 180 were selected and 124 samples were successfully genotyped (68.9%). In total, 45 unique individual foxes were identified and 26 associated with at least one sample which tested positive for E. multilocularis (Em(+)). Estimation of the population size showed the fox population to be between 29 and 34 individuals for a given year and 67 individuals over 4 years. One-third of infected individuals (9/26 Em(+) foxes) deposited 2/3 of the faeces which tested positive for E. multilocularis (36/60 Em(+) stools). Genetic investigation showed a significantly higher average number of multiple stools for females than males, suggesting that the two sexes potentially defecated unequally in the studied area. Three partially overlapping clusters of fox faeces were found, with one cluster concentrating 2/3 of the total E. multilocularis-positive faeces. Based on these findings, we estimated that 12.5 million E. multilocularis eggs were produced during the study period, emphasizing the high contamination level of the environment and the risk of exposure faced by the parasite hosts.

Identification and exploration of a new M2 macrophage marker MTLN in alveolar echinococcosis.

The pathogen of alveolar echinococcosis (AE) is Echinococcus multilocularis (E. multilocularis), which has the characteristics of diffuse infiltration and growth and has a high mortality rate. At present, the role of macrophages in AE infection has attracted more and more attention, but the new biomarkers and polarization mechanisms of macrophages are rarely studied. In this study, CIBERSORT and WGCNA algorithms were used to establish a weighted gene co-expression network, and MTLN was identified as a biological marker of M2-type macrophages, which participated in energy metabolism of macrophages and mediated inflammatory response, but the role of MTLN in AE was not studied. In this study, liver tissue samples from AE patients were collected and immunofluorescence co-localization showed the relationship between MTLN and macrophage distribution. E. multilocularis infected mouse model was established to analyze the expression of MTLN, liver fibrosis, and inflammatory reaction after E. multilocularis infection. The cell experiment simulated the liver microenvironment of E. multilocularis infected human body and analyzed the expression of MTLN by QRT-PCR and western blot in vitro. The data showed that liver fibrosis occurred in AE patients, and MTLN was activated near the focus. After E. multilocularis infected mice, the expression of MTLN increased with time. In the cell experiment, after the antigen of E. multilocularis protoscolex stimulated normal liver cells, the expression of MTLN increased 48 h, at this time, M2 was up-regulated and M1 was down-regulated. Therefore, MTLN may be the key gene to regulate the polarization of M2 macrophages and cause fibrosis.

Risk factors for Echinococcus multilocularis intestinal infections in owned domestic dogs in a North American metropolis (Calgary, Alberta).

Human alveolar echinococcosis is increasingly documented in Alberta, Canada. Its causative agent, Echinococcus multilocularis (Em), can be transmitted to humans by infected dogs. We assessed the prevalence and associated risk factors for Em infections in domestic dogs in Calgary, Alberta, Canada. In this cross-sectional study that coupled collection and assessment of dog feces with a survey on potential risk factors, 13 of 696 (Bayesian true prevalence, 2.4%; 95% CrI: 1.3-4.0%) individual dogs' feces collected during August and September 2012 were qPCR positive for Em. Sequencing two of these cases indicated that both were from the same Em European strain responsible for human infections in Alberta. Likelihood of intestinal Em was 5.6-times higher in hounds than other breeds, 4.6-times higher in dogs leashed at dog parks than those allowed off-leash, 3.1-times higher in dogs often kept in the backyard during spring and summer months than those rarely in the yard, and 3.3-times higher in dogs living in neighbourhoods bordering Bowmont park than those in other areas of Calgary. This situation warrants surveillance of dog infections as a preventative measure to reduce infections in North America.

Genome-wide transcriptome analysis of Echinococcus multilocularis larvae and germinative cell cultures reveals genes involved in parasite stem cell function.

The lethal zoonosis alveolar echinococcosis is caused by tumour-like growth of the metacestode stage of the tapeworm Echinococcus multilocularis within host organs. We previously demonstrated that metacestode proliferation is exclusively driven by somatic stem cells (germinative cells), which are the only mitotically active parasite cells that give rise to all differentiated cell types. The Echinococcus gene repertoire required for germinative cell maintenance and differentiation has not been characterised so far. We herein carried out Illumina sequencing on cDNA from Echinococcus metacestode vesicles, from metacestode tissue depleted of germinative cells, and from Echinococcus primary cell cultures. We identified a set of ~1,180 genes associated with germinative cells, which contained numerous known stem cell markers alongside genes involved in replication, cell cycle regulation, mitosis, meiosis, epigenetic modification, and nucleotide metabolism. Interestingly, we also identified 44 stem cell associated transcription factors that are likely involved in regulating germinative cell differentiation and/or pluripotency. By in situ hybridization and pulse-chase experiments, we also found a new general Echinococcus stem cell marker, EmCIP2Ah, and we provide evidence implying the presence of a slow cycling stem cell sub-population expressing the extracellular matrix factor Emkal1. RNA-Seq analyses on primary cell cultures revealed that metacestode-derived Echinococcus stem cells display an expanded differentiation capability and do not only form differentiated cell types of the metacestode, but also cells expressing genes specific for protoscoleces, adult worms, and oncospheres, including an ortholog of the schistosome praziquantel target, EmTRPMPZQ. Finally, we show that primary cell cultures contain a cell population expressing an ortholog of the tumour necrosis factor α receptor family and that mammalian TNFα accelerates the development of metacestode vesicles from germinative cells. Taken together, our analyses provide a robust and comprehensive characterization of the Echinococcus germinative cell transcriptome, demonstrate expanded differentiation capability of metacestode derived stem cells, and underscore the potential of primary germinative cell cultures to investigate developmental processes of the parasite. These data are relevant for studies into the role of Echinococcus stem cells in parasite development and will facilitate the design of anti-parasitic drugs that specifically act on the parasite germinative cell compartment.

Mitochondrial genetic diversity and phylogenetic relationships of Echinococcus multilocularis in Europe.

The cestode Echinococcus multilocularis is the causative agent of alveolar echinococcosis, a fatal zoonotic parasitic disease of the northern hemisphere. Red foxes are the main reservoir hosts and, likely, the main drivers of the geographic spread of the disease in Europe. Knowledge of genetic relationships among E. multilocularis isolates at a European scale is key to understanding the dispersal characteristics of E. multilocularis. Hence, the present study aimed to describe the genetic diversity of E. multilocularis isolates obtained from different host species in 19 European countries. Based on the analysis of complete nucleotide sequences of the cob, atp6, nad2, nad1 and cox1 mitochondrial genes (4,968 bp), 43 haplotypes were inferred. Four haplotypes represented 62.56 % of the examined isolates (142/227), and one of these four haplotypes was found in each country investigated, except Svalbard, Norway. While the haplotypes from Svalbard were markedly different from all the others, mainland Europe appeared to be dominated by two main clusters, represented by most western, central and eastern European countries, and the Baltic countries and northeastern Poland, respectively. Moreover, one Asian-like haplotype was identified in Latvia and northeastern Poland. To better elucidate the presence of Asian genetic variants of E. multilocularis in Europe, and to obtain a more comprehensive Europe-wide coverage, further studies, including samples from endemic regions not investigated in the present study, especially some eastern European countries, are needed. Further, the present work proposes historical causes that may have contributed to shaping the current genetic variability of E. multilocularis in Europe.

Human alveolar echinococcosis in Slovakia: Epidemiology and genetic diversity of Echinococcus multilocularis, 2000-2023.

Human alveolar echinococcosis (AE) is a serious parasitic disease caused by larval stages of Echinococcus multilocularis. Between January 2000 and October 2023, 137 AE cases were confirmed in Slovakia. The average annual incidence increased from 0.031 per 100,000 inhabitants between 2000 and 2011, to an average of 0.187 since 2012, i.e. about six times. Among patients, 45.3% were men and 54.7% were women; the mean age at the time of diagnosis was 52.8 years. Most cases were diagnosed in the age groups 51-60 years and 61-70 years (33 cases each), and eight patients fell into the age category ≤ 20 years. To better recognize the gene diversity in clinical samples, metacestodes from 21 patients collected between 2013 and 2021 were subjected to DNA sequencing of four mitochondrial genes. Using concatenated sequences of cob (603 bp), nad2 (882 bp) and cox1 (789 bp) gene fragments, 14 isolates (66.7%) were assigned to the European E5 profile of E. multilocularis, two isolates (9.5%) to the E5a subtype, four isolates (19%) to the E4 profile, and one isolate (4.8%) to haplogroup E1/E2. The E5-type profiles and E4 profiles were distributed throughout the country, whereas the E1/E2 profile was found in the patient from western Slovakia. According to the data obtained and GenBank sequences, the E5-type dispersal is so far limited to central-eastern Europe and the variant seems to be indigenous to that region. The admixture with the haplotypes E4 and E1/E2 could have taken place from a historical endemic focus during the fox expansion in the last decades. By employing the nad1 fragment, a typical European haplotype was observed in all 21 resolved Slovak samples. The acceleration in the AE incidence in the last decade suggests the emergence of the disease and the need for further research on human and animal isolates.

Omics-based investigation of pathological liver injury induced by Echinococcus multilocularis infection in mice.

BACKGROUND: Alveolar echinococcosis (AE) can cause severe liver injury and be fatal if left untreated. Currently, there are no effective therapeutic options for AE-induced liver injury. Therefore, by exploring the changes of gene proteins in mice with damaged liver, we attempted to identify the key molecules of liver damage, and provide data that will enable the development of drugs targeting hepatic AE. METHODS: BALB/c mice were inoculated with protoscoleces via the hepatic portal vein. Three months later, B-ultrasound examination and Hematoxylin-eosin (H&E) staining were used to confirm liver damage in mice. RNA sequencing and Liquid chromatography-mass spectrometry (LC-MS) were used to screen differentially expressed molecules associated with liver damage through bioinformatics, and Quantitative Real-Time PCR (qRT-PCR) was used to verify their expression. RESULTS: B-ultrasound examination showed liver lesions in the infected group, and H&E staining showed liver inflammation, fibrosis and liver necrosis. RNA sequencing and LC-MS results showed changes in the levels of more than 1000 genes and proteins, with upregulation of immune and inflammation pathways. By contrast, the downregulated genes and proteins were mostly involved in various metabolic reactions. Correlation analysis was conducted between the transcriptome data and proteome data. The results revealed 240 differentially expressed genes, of which 192 were upregulated, and 48 were downregulated. Many of these genes were involved in metabolic reactions, such as Catalase (Cat), fatty acid synthase (Fasn), and IL-16 genes, which may have relevance to liver injury. The results of qRT-PCR were consistent with those of bioinformatics analysis. CONCLUSIONS: The mechanisms of liver injury in mice infected with Echinococcus multilocularis are complex, involving abnormal metabolism, oxidative stress, inflammatory response, and many other factors. This study provides the data for preliminary exploration for the development of targeted therapies against AE.

Genetic diversity of Echinococcus multilocularis specimens isolated from Belgian patients with alveolar echinococcosis using EmsB microsatellites analysis.

The genetic diversity of Echinococcus multilocularis (E. multilocularis) specimens isolated from patients with alveolar echinococcosis (AE), is a major field of investigation to correlate with sources of infection, clinical manifestations and prognosis of the disease. Molecular markers able to distinguish samples are commonly used worldwide, including the EmsB microsatellite. Here, we report the use of the EmsB microsatellite polymorphism data mining for the retrospective typing of Belgian specimens of E. multilocularis infecting humans. A total of 18 samples from 16 AE patients treated between 2006 and 2021 were analyzed through the EmsB polymorphism. Classification of specimens was performed through a dendrogram construction in order to compare the similarity among Belgian samples, some human referenced specimens on the EWET database (EmsB Website for the Echinococcus Typing) and previously published EmsB profiles from red foxes circulating in/near Belgium. According to a comparison with human European specimens previously genotyped in profiles, the 18 Belgian ones were classified into three EmsB profiles. Four specimens could not be assigned to an already known profile but some are near to EWET referenced samples. This study also highlights that some specimens share the same EmsB profile with profiles characterized in red foxes from north Belgium, the Netherlands, Luxembourg and French department near to the Belgian border. Furthermore, Belgian specimens present a genetic diversity and include one profile that don't share similarities with the ones referenced in the EWET database. However, at this geographical scale, there is no clear correlation between EmsB profiles and geographical location. Further studies including additional clinical samples and isolates from foxes and rodents of south Belgium are necessary to better understand the spatial and temporal circumstances of human infections but also a potential correlation between EmsB profiles and parasite virulence.

NOVEL REPORT OF THE EUROPEAN VARIANT OF ECHINOCOCCUS MULTILOCULARIS IN COYOTES (CANIS LATRANS) IN NEW YORK STATE.

Echinococcus multilocularis is a zoonotic cestode that can infect wildlife, domestic animals, and humans. In humans, infection with the larval stage of the parasite causes the disease alveolar echinococcosis, which can be fatal if left untreated. Surveillance for the parasite in New York State occurred during the 2021-2022 coyote (Canis latrans) hunting season. Fecal samples and the gastrointestinal tracts (GIT) from 43 coyote carcasses were collected from hunters and trappers across 8 counties. Fecal samples were screened for E. multilocularis DNA using a multiplex PCR. Three samples tested positive for E. multilocularis DNA. Subsequently, adult cestodes were collected from GIT samples using the sedimentation, filtration, and counting technique. Phylogenetic analysis of DNA sequences from the nad2 and cob genes from individual worms indicated these New York sequences cluster with E. multilocularis sequences from Europe. This is the first report of adult E. multilocularis cestodes in New York State, as well as the first detection of the European haplotype of E. multilocularis in wildlife in the northeastern United States.

Infection of sheep by Echinococcus multilocularis in Gansu, China: evidence from mitochondrial and nuclear DNA analysis.

BACKGROUND: In the normal life cycle of the parasite (Echinococcus multilocularis) that causes alveolar echinococcosis, domestic and wild carnivores act as definitive hosts, and rodents act as intermediate hosts. The presented study contributes to the research on the distribution and transmission pattern of E. multilocularis in China having identified sheep as an unusual intermediate host taking part in the domestic transmission of alveolar echinococcosis in Gansu Province, China. METHODS: From 2020 to 2021, nine whitish different cyst-like were collected from the liver of sheep in Gansu Province for examination. A near complete mitochondrial (mt) genome and selected nuclear genes were amplified from the cyst-like lesion for identification. To confirm the status of the specimen, comparative analysis with reference sequences, phylogenetic analysis, and network analysis were performed. RESULTS: The isolates displayed ≥ 98.87% similarity to E. multilocularis NADH dehydrogenase sub-unit 1 (nad1) (894 bp) reference sequences deposited in GenBank. Furthermore, amplification of the nad4 and nad2 genes also confirmed all nine samples as E. multilocularis with > 99.30% similarity. Additionally, three nuclear genes, pepck (1545 bp), elp-exons VII and VIII (566 bp), and elp-exon IX (256 bp), were successfully amplified and sequenced for one of the isolates with 98.42% similarity, confirming the isolates were correctly identified as E. multilocularis. Network analysis also correctly placed the isolates with other E. multilocularis. CONCLUSIONS: As a result of the discovery of E. multilocularis in an unusual intermediate host, which is considered to have the highest zoonotic potential, the result clearly demonstrated the necessity for expanded surveillance in the area.

Detection of Echinococcus multilocularis in repurposed environmental DNA samples from river water.

Environmental DNA (eDNA) is an increasingly popular tool in biological and ecological studies. As a biproduct of its increasing use, large number of eDNA samples are being collected and stored, that potentially contain information of many non-target species. One potential use for these eDNA samples is a surveillance and early detection of pathogens and parasites that are otherwise difficult to detect. Echinococcus multilocularis is such a parasite with serious zoonotic concern, and whose range has been expanding. If eDNA samples from various studies can be repurposed in detecting the parasite, it can significantly reduce the costs and efforts in surveillance and early detection of the parasite. We designed and tested a new set of primer-probe for detecting E. multilocularis mitochondrial DNA in environmental medium. Using this primer-probe set, we conducted real-time PCR on repurposed eDNA samples collected from three streams flowing through an area of Japan endemic to the parasite. We detected the DNA of E. multilocularis in one of the 128 samples (0.78%). The discovery suggests that while detecting E. multilocularis using eDNA samples is possible, the rate of detection appear to be very low. However, given the naturally low prevalence of the parasite among wild hosts in endemic areas, the repurposed eDNAs may still be a valid option for surveillance in newly introduced areas with the reduced cost and efforts. Further work is needed to assess and improve the effectiveness of using eDNA for detection of E. multilocularis.

Novel molecular diagnostic (PCR) diagnosis and outcome of intestinal Echinococcus multilocularis in a dog from western Canada.

OBJECTIVE: To describe the novel PCR diagnosis and outcome of intestinal Echinococcus multilocularis in a dog. ANIMAL: A 13-month-old female intact dog with naturally occurring intestinal E multilocularis. CLINICAL PRESENTATION, PROGRESSION, AND PROCEDURES: The 13-month-old dog initially presented with a reduced appetite and weight loss and then developed hematochezia. The clinical history included a lack of endoparasite preventive care (fecal testing, deworming), exposure to coyotes, fox, sheep, and rodents and the dog had intermittently been fed a raw food diet. Physical examination revealed a thin dog, with a 2/9 body condition score, that was otherwise clinically unremarkable. A fecal sample was submitted for screening for gastrointestinal parasites as part of an infectious disease assessment. The fecal PCR test reported detection of E multilocularis. This result was sequenced as the European haplotype E3/E4. Centrifugal flotation (same sample) did not detect taeniid eggs. TREATMENT AND OUTCOME: The dog was treated with metronidazole, maropitant, and milbemycin oxime/praziquantel. Clinical improvement was noted within 48 hours. No DNA of E multilocularis was detected in a fecal sample collected approximately 10 days after treatment. The dog's owner was advised to provide monthly deworming (praziquantel) for all dogs on the property and to contact their human health-care provider due to potential zoonotic exposure risk. CLINICAL RELEVANCE: Increasing detection of E multilocularis is occurring in dogs in Canada and the US. Alveolar echinococcosis can cause severe disease in dogs and humans. Fecal PCR detection and surveillance may alert practitioners to canine intestinal cases and allow dogs to serve as sentinels for human exposure risk.

Complete mitochondrial exploration of Echinococcus multilocularis from French alveolar echinococcosis patients.

Alveolar echinococcosis (AE) is a parasitosis that is expanding worldwide, including in Europe. The development of genotypic markers is essential to follow its spatiotemporal evolution. Sequencing of the commonly used mitochondrial genes cob, cox1, and nad2 shows low discriminatory power, and analysis of the microsatellite marker EmsB does not allow nucleotide sequence analysis. We aimed to develop a new method for the genotyping of Echinococcus multilocularis based on whole mitochondrial genome (mitogenome) sequencing, to determine the genetic diversity among 30 human visceral samples from French patients, and compare this method with those currently in use. Sequencing of the whole mitochondrial genome was carried out after amplification by PCR, using one uniplex and two multiplex reactions to cover the 13,738 bp of the mitogenome, combined with Illumina technology. Thirty complete mitogenome sequences were obtained from AE lesions. One showed strong identity with Asian genotypes (99.98% identity) in a patient who had travelled to China. The other 29 mitogenomes could be differentiated into 13 haplotypes, showing higher haplotype and nucleotide diversity than when using the cob, cox1, and nad2 gene sequences alone. The mitochondrial genotyping data and EmsB profiles did not overlap, probably because one method uses the mitochondrial genome and the other the nuclear genome. The pairwise fixation index (Fst) value between individuals living inside and those living outside the endemic area was high (Fst = 0.222, P = 0.002). This is consistent with the hypothesis of an expansion from historical endemic areas to peripheral regions.