Detection of Echinococcus multilocularis in carnivores in Razavi Khorasan province, Iran using mitochondrial DNA

Background

Echinococcus multilocularis is the source of alveolar echinococcosis, a potentially fatal zoonotic disease. This investigation assessed the presence of E. multilocularis infection in definitive hosts in the Chenaran region of Razavi Khorasan Province, northeastern Iran.

Methodology/Principal Findings

Fecal samples from 77 domestic and stray dogs and 14 wild carnivores were examined using the flotation/sieving method followed by multiplex PCR of mitochondrial genes. The intestinal scraping technique (IST) and the sedimentation and counting technique (SCT) revealed adult Echinococcus in the intestines of five of 10 jackals and of the single wolf examined. Three jackals were infected only with E. multilocularis but two, and the wolf, were infected with both E. multilocularis and E. granulosus. Multiplex PCR revealed E. multilocularis, E. granulosus, and Taenia spp. in 19, 24, and 28 fecal samples, respectively. Echinococcus multilocularis infection was detected in the feces of all wild carnivores sampled including nine jackals, three foxes, one wolf, one hyena, and five dogs (6.5%). Echinococcus granulosus was found in the fecal samples of 16.9% of dogs, 66.7% of jackals, and all of the foxes, the wolf, and the hyena. The feces of 16 (21.8%) dogs, 7 of 9 (77.8%) jackals, and all three foxes, one wolf and one hyena were infected with Taenia spp.

Conclusions/Significance

The prevalence of E. multilocularis in wild carnivores of rural areas of the Chenaran region is high, indicating that the life cycle is being maintained in northeastern Iran with the red fox, jackal, wolf, hyena, and dog as definitive hosts.     

Natural Infection of the Ground Squirrel (Spermophilus spp.) with Echinococcus granulosus in China

Background

Echinococcus granulosus is usually transmitted between canid definitive hosts and ungulate intermediate hosts.

Methodology/Principal Findings

Lesions found in the livers of ground squirrels, Spermophilus dauricus/alashanicus, trapped in Ningxia Hui Autonomous Region, an area in China co-endemic for both E. granulosus and E. multilocularis, were subjected to molecular genotyping for Echinococcus spp. DNA. One of the lesions was shown to be caused by E. granulosus and subsequently by histology to contain viable protoscoleces.

Conclusions/Significance

This is the first report of a natural infection of the ground squirrel with E. granulosus. This does not provide definitive proof of a cycle involving ground squirrels and dogs or foxes, but it is clear that there is active E. granulosus transmission occurring in this area, despite a recent past decline in the dog population in southern Ningxia.     

Drivers of Echinococcus multilocularis Transmission in China: Small Mammal Diversity,Landscape or Climate?

Background

Human alveolar echinococcocosis (AE) is a highly pathogenic zoonotic disease caused by the larval stage of the cestode E. multilocularis. Its life-cycle includes more than 40 species of small mammal intermediate hosts. Therefore, host biodiversity losses could be expected to alter transmission. Climate may also have possible impacts on E. multilocularis egg survival. We examined the distribution of human AE across two spatial scales, (i) for continental China and (ii) over the eastern edge of the Tibetan plateau. We tested the hypotheses that human disease distribution can be explained by either the biodiversity of small mammal intermediate host species, or by environmental factors such as climate or landscape characteristics.

Methodology/findings

The distributions of 274 small mammal species were mapped to 967 point locations on a grid covering continental China. Land cover, elevation, monthly rainfall and temperature were mapped using remotely sensed imagery and compared to the distribution of human AE disease at continental scale and over the eastern Tibetan plateau. Infection status of 17,589 people screened by abdominal ultrasound in 2002–2008 in 94 villages of Tibetan areas of western Sichuan and Qinghai provinces was analyzed using generalized additive mixed models and related to epidemiological and environmental covariates. We found that human AE was not directly correlated with small mammal reservoir host species richness, but rather was spatially correlated with landscape features and climate which could confirm and predict human disease hotspots over a 200,000 km² region.

Conclusions/Significance

E. multilocularis transmission and resultant human disease risk was better predicted from landscape features that could support increases of small mammal host species prone to population outbreaks, rather than host species richness. We anticipate that our study may be a starting point for further research wherein landscape management could be used to predict human disease risk and for controlling this zoonotic helminthic.     

Landscape composition and spatial prediction of alveolar echinococcosis in southern Ningxia, China

Background

Alveolar echinococcosis (AE) presents a serious public health challenge within China. Mass screening ultrasound surveys can detect pre-symptomatic AE, but targeting areas identified from hospital records is inefficient regarding AE. Prediction of undetected or emerging hotspots would increase detection rates. Voles and lemmings of the subfamily Arvicolinae are important intermediate hosts in sylvatic transmission systems. Their populations reach high densities in productive grasslands where food and cover are abundant. Habitat availability is thought to affect arvicoline population dynamic patterns and definitive host–intermediate host interactions. Arvicoline habitat correlates with AE prevalence in Western Europe and southern Gansu Province, China.

Methods and Findings

Xiji County, Ningxia Hui Autonomous Region, borders southern Gansu. The aims of this study were to map AE prevalence across Xiji and test arvicoline habitat as a predictor. Land cover was mapped using remotely sensed (Landsat) imagery. Infection status of 3,205 individuals screened in 2002–2003 was related, using generalised additive mixed models, to covariates: gender; farming; ethnicity; dog ownership; water source; and areal cover of mountain pasture and lowland pasture. A Markov random field modelled additional spatial variation and uncertainty. Mountain pasture and lowland pasture were associated with below and above average AE prevalence, respectively.

Conclusions

Low values of the normalised difference vegetation index indicated sub-optimality of lowland pasture for grassland arvicolines. Unlike other known endemic areas, grassland arvicolines probably did not provide the principal reservoir for Echinococcus multilocularis in Xiji. This result is consistent with recent small mammal surveys reporting low arvicoline densities and high densities of hamsters, pikas and jerboas, all suitable intermediate hosts for E. multilocularis, in reforested lowland pasture. The risk of re-emergence is discussed. We recommend extending monitoring to: southern Haiyuan County, where predicted prevalence was high; southern Xiji County, where prediction uncertainty was high; and monitoring small mammal community dynamics and the infection status of dogs.     

Excretory/secretory-products of Echinococcus multilocularis larvae induce apoptosis and tolerogenic properties in dendritic cells in vitro

Background

Alveolar echinococcosis, caused by Echinococcus multilocularis larvae, is a chronic disease associated with considerable modulation of the host immune response. Dendritic cells (DC) are key effectors in shaping the immune response and among the first cells encountered by the parasite during an infection. Although it is assumed that E.multilocularis, by excretory/secretory (E/S)-products, specifically affects DC to deviate immune responses, little information is available on the molecular nature of respective E/S-products and their mode of action.

Methodology/Principal Findings

We established cultivation systems for exposing DC to live material from early (oncosphere), chronic (metacestode) and late (protoscolex) infectious stages. When co-incubated with Echinococcus primary cells, representing the invading oncosphere, or metacestode vesicles, a significant proportion of DC underwent apoptosis and the surviving DC failed to mature. In contrast, DC exposed to protoscoleces upregulated maturation markers and did not undergo apoptosis. After pre-incubation with primary cells and metacestode vesicles, DC showed a strongly impaired ability to be activated by the TLR ligand LPS, which was not observed in DC pre-treated with protoscolex E/S-products. While none of the larvae induced the secretion of pro-inflammatory IL-12p70, the production of immunosuppressive IL-10 was elevated in response to primary cell E/S-products. Finally, upon incubation with DC and naïve T-cells, E/S-products from metacestode vesicles led to a significant expansion of Foxp3+ T cells in vitro.

Conclusions

This is the first report on the induction of apoptosis in DC by cestode E/S-products. Our data indicate that the early infective stage of E. multilocularis is a strong inducer of tolerance in DC, which is most probably important for generating an immunosuppressive environment at an infection phase in which the parasite is highly vulnerable to host attacks. The induction of CD4+CD25+Foxp3+ T cells through metacestode E/S-products suggests that these cells fulfill an important role for parasite persistence during chronic echinococcosis.     

A Systematic Review of the Epidemiology of Echinococcosis in Domestic and Wild Animals

Background

Human echinococcosis is a neglected zoonosis caused by parasites of the genus Echinococcus. The most frequent clinical forms of echinococcosis, cystic echinococcosis (CE) and alveolar echinococcosis (AE), are responsible for a substantial health and economic burden, particularly to low-income societies. Quantitative epidemiology can provide important information to improve the understanding of parasite transmission and hence is an important part of efforts to control this disease. The purpose of this review is to give an insight on factors associated with echinococcosis in animal hosts by summarising significant results reported from epidemiological studies identified through a systematic search.

Methodology and Principal Findings

The systematic search was conducted mainly in electronic databases but a few additional records were obtained from other sources. Retrieved entries were examined in order to identify available peer-reviewed epidemiological studies that found significant risk factors for infection using associative statistical methods. One hundred studies met the eligibility criteria and were suitable for data extraction. Epidemiological factors associated with increased risk of E. granulosus infection in dogs included feeding with raw viscera, possibility of scavenging dead animals, lack of anthelmintic treatment and owners'' poor health education and indicators of poverty. Key factors associated with E. granulosus infection in intermediate hosts were related to the hosts'' age and the intensity of environmental contamination with parasite eggs. E. multilocularis transmission dynamics in animal hosts depended on the interaction of several ecological factors, such as hosts'' population densities, host-prey interactions, landscape characteristics, climate conditions and human-related activities.

Conclusions/Significance

Results derived from epidemiological studies provide a better understanding of the behavioural, biological and ecological factors involved in the transmission of this parasite and hence can aid in the design of more effective control strategies.     

EmTIP,a T-Cell Immunomodulatory Protein Secreted by the Tapeworm Echinococcus multilocularis Is Important for Early Metacestode Development

Background

Alveolar echinococcosis (AE), caused by the metacestode of the tapeworm Echinococcus multilocularis, is a lethal zoonosis associated with host immunomodulation. T helper cells are instrumental to control the disease in the host. Whereas Th1 cells can restrict parasite proliferation, Th2 immune responses are associated with parasite proliferation. Although the early phase of host colonization by E. multilocularis is dominated by a potentially parasitocidal Th1 immune response, the molecular basis of this response is unknown.

Principal Findings

We describe EmTIP, an E. multilocularis homologue of the human T-cell immunomodulatory protein, TIP. By immunohistochemistry we show EmTIP localization to the intercellular space within parasite larvae. Immunoprecipitation and Western blot experiments revealed the presence of EmTIP in the excretory/secretory (E/S) products of parasite primary cell cultures, representing the early developing metacestode, but not in those of mature metacestode vesicles. Using an in vitro T-cell stimulation assay, we found that primary cell E/S products promoted interferon (IFN)-γ release by murine CD4+ T-cells, whereas metacestode E/S products did not. IFN-γ release by T-cells exposed to parasite products was abrogated by an anti-EmTIP antibody. When recombinantly expressed, EmTIP promoted IFN-γ release by CD4+ T-cells in vitro. After incubation with anti-EmTIP antibody, primary cells showed an impaired ability to proliferate and to form metacestode vesicles in vitro.

Conclusions

We provide for the first time a possible explanation for the early Th1 response observed during E. multilocularis infections. Our data indicate that parasite primary cells release a T-cell immunomodulatory protein, EmTIP, capable of promoting IFN-γ release by CD4+ T-cells, which is probably driving or supporting the onset of the early Th1 response during AE. The impairment of primary cell proliferation and the inhibition of metacestode vesicle formation by anti-EmTIP antibodies suggest that this factor fulfills an important role in early E. multilocularis development within the intermediate host.     

Synthesising 30 Years of Mathematical Modelling of Echinococcus Transmission

Background

Echinococcosis is a complex zoonosis that has domestic and sylvatic lifecycles, and a range of different intermediate and definitive host species. The complexities of its transmission and the sparse evidence on the effectiveness of control strategies in diverse settings provide significant challenges for the design of effective public health policy against this disease. Mathematical modelling is a useful tool for simulating control packages under locally specific transmission conditions to inform optimal timing and frequency of phased interventions for cost-effective control of echinococcosis. The aims of this review of 30 years of Echinococcus modelling were to discern the epidemiological mechanisms underpinning models of Echinococcus granulosus and E. multilocularis transmission and to establish the need to include a human transmission component in such models.

Methodology/Principal Findings

A search was conducted of all relevant articles published up until July 2012, identified from the PubMED, Web of Knowledge and Medline databases and review of bibliographies of selected papers. Papers eligible for inclusion were those describing the design of a new model, or modification of an existing mathematical model of E. granulosus or E. multilocularis transmission. A total of 13 eligible papers were identified, five of which described mathematical models of E. granulosus and eight that described E. multilocularis transmission. These models varied primarily on the basis of six key mechanisms that all have the capacity to modulate model dynamics, qualitatively affecting projections. These are: 1) the inclusion of a ‘latent’ class and/or time delay from host exposure to infectiousness; 2) an age structure for animal hosts; 3) the presence of density-dependent constraints; 4) accounting for seasonality; 5) stochastic parameters; and 6) inclusion of spatial and risk structures.

Conclusions/Significance

This review discusses the conditions under which these mechanisms may be important for inclusion in models of Echinococcus transmission and proposes recommendations for the design of dynamic human models of transmission. Accounting for the dynamic behaviour of the Echinococcus parasites in humans will be key to predicting changes in the disease burden over time and to simulate control strategies that optimise public health impact.     

Serodiagnosis of Echinococcus spp. Infection: Explorative Selection of Diagnostic Antigens by Peptide Microarray

Background

Production of native antigens for serodiagnosis of helminthic infections is laborious and hampered by batch-to-batch variation. For serodiagnosis of echinococcosis, especially cystic disease, most screening tests rely on crude or purified Echinococcus granulosus hydatid cyst fluid. To resolve limitations associated with native antigens in serological tests, the use of standardized and highly pure antigens produced by chemical synthesis offers considerable advantages, provided appropriate diagnostic sensitivity and specificity is achieved.

Methodology/Principal Findings

Making use of the growing collection of genomic and proteomic data, we applied a set of bioinformatic selection criteria to a collection of protein sequences including conceptually translated nucleotide sequence data of two related tapeworms, Echinococcus multilocularis and Echinococcus granulosus. Our approach targeted alpha-helical coiled-coils and intrinsically unstructured regions of parasite proteins potentially exposed to the host immune system. From 6 proteins of E. multilocularis and 5 proteins of E. granulosus, 45 peptides between 24 and 30 amino acids in length were designed. These peptides were chemically synthesized, spotted on microarrays and screened for reactivity with sera from infected humans. Peptides reacting above the cut-off were validated in enzyme-linked immunosorbent assays (ELISA). Peptides identified failed to differentiate between E. multilocularis and E. granulosus infection. The peptide performing best reached 57% sensitivity and 94% specificity. This candidate derived from Echinococcus multilocularis antigen B8/1 and showed strong reactivity to sera from patients infected either with E. multilocularis or E. granulosus.

Conclusions/Significance

This study provides proof of principle for the discovery of diagnostically relevant peptides by bioinformatic selection complemented with screening on a high-throughput microarray platform. Our data showed that a single peptide cannot provide sufficient diagnostic sensitivity whereas pooling several peptide antigens improved sensitivity; thus combinations of several peptides may lead the way to new diagnostic tests that replace, or at least complement conventional immunodiagnosis of echinococcosis. Our strategy could prove useful for diagnostic developments in other pathogens.     

Latent Microsporidiosis Caused by Encephalitozoon cuniculi in Immunocompetent Hosts: A Murine Model Demonstrating the Ineffectiveness of the Immune System and Treatment with Albendazole

Background

Microsporidia are obligate intracellular parasites causing severe infections with lethal outcome in immunocompromised hosts. However, these pathogens are more frequently reported as latent infections in immunocompetent individuals and raises questions about the potential risk of reactivation following induced immunosuppression.

Aims

To evaluate the possibility latent microsporidiosis, efficacy or albendazole, and reactivation, the authors monitored the course of E. cuniculi infection in immunocompetent BALB/c mice and immunodeficient SCID mice using molecular methods.

Methods

Mice were per orally infected with 10⁷ spores of E. cuniculi. Selected groups were treated with albendazole, re-infected or chemically immunosuppressed by dexamethasone. The presence of microsporidia in the host’s organs and feces were determined using PCR methods. Changes in numbers of lymphocytes in blood and in spleen after induction of immunosuppression were confirmed using flow cytometry analysis.

Results

Whereas E. cuniculi caused lethal microsporidiosis in SCID mice, the infection in BABL/c mice remained asymptomatic despite parasite dissemination into many organs during the acute infection phase. Albendazole treatment led to microsporidia elimination from organs in BALB/c mice. In SCID mice, however, only a temporary reduction in number of affected organs was observed and infection re-established post-treatment. Dexamethasone treatment resulted in a chronic microsporidia infection disseminating into most organs in BALB/c mice. Although the presence of E. cuniculi in organs of albendazole- treated mice was undetectable by PCR, it was striking that infection was reactivated by immunosuppression treatment.

Conclusion

Our results demonstrated that microsporidia can successfully survive in organs of immunocompetent hosts and are able to reactivate from undetectable levels and spread within these hosts after induction of immunosuppression. These findings stress the danger of latent microsporidiosis as a life-threatening risk factor especially for individuals undergoing chemotherapy and in transplant recipients of organs originating from infected donors.     

Genetic Diversity of the Cestode Echinococcus multilocularis in Red Foxes at a Continental Scale in Europe

Background

Alveolar echinococcosis (AE) is a severe helminth disease affecting humans, which is caused by the fox tapeworm Echinococcus multilocularis. AE represents a serious public health issue in larger regions of China, Siberia, and other regions in Asia. In Europe, a significant increase in prevalence since the 1990s is not only affecting the historically documented endemic area north of the Alps but more recently also neighbouring regions previously not known to be endemic. The genetic diversity of the parasite population and respective distribution in Europe have now been investigated in view of generating a fine-tuned map of parasite variants occurring in Europe. This approach may serve as a model to study the parasite at a worldwide level.

Methodology/Principal Findings

The genetic diversity of E. multilocularis was assessed based upon the tandemly repeated microsatellite marker EmsB in association with matching fox host geographical positions. Our study demonstrated a higher genetic diversity in the endemic areas north of the Alps when compared to other areas.

Conclusions/Significance

The study of the spatial distribution of E. multilocularis in Europe, based on 32 genetic clusters, suggests that Europe can be considered as a unique global focus of E. multilocularis, which can be schematically drawn as a central core located in Switzerland and Jura Swabe flanked by neighbouring regions where the parasite exhibits a lower genetic diversity. The transmission of the parasite into peripheral regions is governed by a “mainland–island” system. Moreover, the presence of similar genetic profiles in both zones indicated a founder event.     

Time course of gene expression profiling in the liver of experimental mice infected with Echinococcus multilocularis

Background

Alveolar echinococcosis (AE) is a severe chronic parasitic disease which behaves like a slow-growing liver cancer. Clinical observations suggest that the parasite, Echinococcus multilocularis (E. multilocularis) influences liver homeostasis and hepatic cell metabolism. However, this has never been analyzed during the time course of infection in the common model of secondary echinococcosis in experimental mice.

Methodology/Principal Findings

Gene expression profiles were assessed using DNA microarray analysis, 1, 2, 3 and 6 months after injection of E. multilocularis metacestode in the liver of susceptible mice. Data were collected at different time points to monitor the dynamic behavior of gene expression. 557 differentially expressed genes were identified at one or more time points, including 351 up-regulated and 228 down-regulated genes. Time-course analysis indicated, at the initial stage of E. multilocularis infection (month 1–2), that most of up-regulated pathways were related to immune processes and cell trafficking such as chemokine-, mitogen-activated protein kinase (MAPK) signaling, and down-regulated pathways were related to xenobiotic metabolism; at the middle stage (month 3), MAPK signaling pathway was maintained and peroxisome proliferator-activated receptor (PPAR) signaling pathway emerged; at the late stage (month 6), most of up-regulated pathways were related to PPAR signaling pathway, complement and coagulation cascades, while down-regulated pathways were related to metabolism of xenobiotics by cytochrome P450. Quantitative RT-PCR analysis of a random selection of 19 genes confirmed the reliability of the microarray data. Immunohistochemistry analysis showed that proliferating cell nuclear antigen (PCNA) was increased in the liver of E. multilocularis infected mice from 2 months to 6 months.

Conclusions

E. multilocularis metacestode definitely exerts a deep influence on liver homeostasis, by modifying a number of gene expression and metabolic pathways. It especially promotes hepatic cell proliferation, as evidenced by the increased PCNA constantly found in all the experimental time-points we studied and by an increased gene expression of key metabolic pathways.     

Host-specific races in the holoparasitic angiosperm Orobanche minor: implications for speciation in parasitic plants

Background and Aims

Orobanche minor is a root-holoparasitic angiosperm that attacks a wide range of host species, including a number of commonly cultivated crops. The extent to which genetic divergence among natural populations of O. minor is influenced by host specificity has not been determined previously. Here, the host specificity of natural populations of O. minor is quantified for the first time, and evidence that this species may comprise distinct physiological races is provided.

Methods

A tripartite approach was used to examine the physiological basis for the divergence of populations occurring on different hosts: (1) host–parasite interactions were cultivated in rhizotron bioassays in order to quantify the early stages of the infection and establishment processes; (2) using reciprocal-infection experiments, parasite races were cultivated on their natural and alien hosts, and their fitness determined in terms of biomass; and (3) the anatomy of the host–parasite interface was investigated using histochemical techniques, with a view to comparing the infection process on different hosts.

Key Results

Races occurring naturally on red clover (Trifolium pratense) and sea carrot (Daucus carota ssp. gummifer) showed distinct patterns of host specificity: parasites cultivated in cross-infection studies showed a higher fitness on their natural hosts, suggesting that races show local adaptation to specific hosts. In addition, histological evidence suggests that clover and carrot roots vary in their responses to infection. Different root anatomy and responses to infection may underpin a physiological basis for host specificity.

Conclusions

It is speculated that host specificity may isolate races of Orobanche on different hosts, accelerating divergence and ultimately speciation in this genus. The rapid life cycle and broad host range of O. minor make this species an ideal model with which to study the interactions of parasitic plants with their host associates.Key words: Parasitic plant, Orobanche, speciation, divergence, host-specificity, host-specific races     

Spatial heterogeneity and temporal variations in Echinococcus multilocularis infections in wild hosts in a North American urban setting

Echinococcus multilocularis, the causative agent of human alveolar echinococcosis, has the potential to circulate in urban areas where wild host populations and humans coexist. The spatial and temporal distribution of infection in wild hosts locally affects the risk of transmission to humans. We investigated the spatial and temporal patterns of E. multilocularis infection in coyotes and rodent intermediate hosts within the city of Calgary, Canada, and the association between spatial variations in coyote infection and the relative composition of small mammal assemblages. Infection by E. multilocularis was examined in small mammals and coyote faeces collected monthly in five city parks from June 2012 to June 2013. Coyote faeces were analysed using a ZnCl₂ centrifugation and sedimentation protocol. Infection in intermediate hosts was assessed through lethal trapping and post-mortem analysis. Parasite eggs and metacestodes were morphologically identified and molecularly confirmed through species-specific PCR assays. Of 982 small mammals captured, infection was detected in 2/305 (0.66%) deer mice (Peromyscus maniculatus), 2/267 (0.75%) meadow voles (Microtus pennsylvanicus), and 1/71 (1.41%) southern red backed voles (Myodes gapperi). Overall faecal prevalence in coyotes was 21.42% (n = 385) and varied across sites, ranging from 5.34% to 61.48%. Differences in coyote faecal prevalence across sites were consistent with local variations in the relative abundance of intermediate hosts within the small mammal assemblages. Infections peaked in intermediate hosts during autumn (0.68%) and winter (3.33%), and in coyotes during spring (43.47%). Peaks of infections in coyote faeces up to 83.8% in autumn were detected in a hyper-endemic area. To the best of our knowledge, our findings represent the first evidence of a sylvatic life-cycle of E. multilocularis in a North American urban setting, and provide new insights into the complexity of the parasite transmission ecology.     

Combining information from surveys of several species to estimate the probability of freedom from <Emphasis Type="Italic">Echinococcus multilocularis</Emphasis> in Sweden,Finland and mainland Norway

Background  

The fox tapeworm Echinococcus multilocularis has foxes and other canids as definitive host and rodents as intermediate hosts. However, most mammals can be accidental intermediate hosts and the larval stage may cause serious disease in humans. The parasite has never been detected in Sweden, Finland and mainland Norway. All three countries require currently an anthelminthic treatment for dogs and cats prior to entry in order to prevent introduction of the parasite. Documentation of freedom from E. multilocularis is necessary for justification of the present import requirements.