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1.  A semi-automated magnetic capture probe based DNA extraction and real-time PCR method applied in the Swedish surveillance of Echinococcus multilocularis in red fox (Vulpes vulpes) faecal samples 
Parasites & Vectors  2014;7(1):583.
Background
Following the first finding of Echinococcus multilocularis in Sweden in 2011, 2985 red foxes (Vulpes vulpes) were analysed by the segmental sedimentation and counting technique. This is a labour intensive method and requires handling of the whole carcass of the fox, resulting in a costly analysis. In an effort to reduce the cost of labour and sample handling, an alternative method has been developed. The method is sensitive and partially automated for detection of E. multilocularis in faecal samples. The method has been used in the Swedish E. multilocularis monitoring program for 2012–2013 on more than 2000 faecal samples.
Methods
We describe a new semi-automated magnetic capture probe DNA extraction method and real time hydrolysis probe polymerase chain reaction assay (MC-PCR) for the detection of E. multilocularis DNA in faecal samples from red fox. The diagnostic sensitivity was determined by validating the new method against the sedimentation and counting technique in fox samples collected in Switzerland where E. multilocularis is highly endemic.
Results
Of 177 foxes analysed by the sedimentation and counting technique, E. multilocularis was detected in 93 animals. Eighty-two (88%, 95% C.I 79.8-93.9) of these were positive in the MC-PCR. In foxes with more than 100 worms, the MC-PCR was positive in 44 out of 46 (95.7%) cases. The two MC-PCR negative samples originated from foxes with only immature E. multilocularis worms. In foxes with 100 worms or less, (n = 47), 38 (80.9%) were positive in the MC-PCR.
The diagnostic specificity of the MC-PCR was evaluated using fox scats collected within the Swedish screening. Of 2158 samples analysed, two were positive. This implies that the specificity is at least 99.9% (C.I. = 99.7 -100).
Conclusions
The MC-PCR proved to have a high sensitivity and a very high specificity. The test is partially automated but also possible to perform manually if desired. The test is well suited for nationwide E. multilocularis surveillance programs where sampling of fox scats is done to reduce the costs for sampling and where a test with a high sensitivity and a very high specificity is needed.
doi:10.1186/s13071-014-0583-6
PMCID: PMC4282741  PMID: 25522844
Echinococcus multilocularis; Surveillance; Red fox; Diagnostic method; Magnetic capture; Real-time PCR; Faecal samples
2.  First case of peritoneal cysticercosis in a non-human primate host (Macaca tonkeana) due to Taenia martis 
Parasites & Vectors  2014;7(1):422.
Background
Infections with larval stages (metacestodes) of a variety of taeniid species have been described in primates, including humans, with partial to severe clinical consequences. Taenia martis is a tapeworm of mustelids, and martens are mainly their definitive hosts in Central Europe. In the rodent intermediate host cysticerci develop in the pleural and peritoneal cavities. The present report describes a case of T. martis peritoneal cysticercosis in a Tonkean macaque.
Findings
An abdominal mass was detected in a 3-year-old male Tonkean macaque (Macaca tonkeana) born and raised in a primate colony in France. Examination of the mass after laparotomy showed numerous vesicles identified as cysticerci of T. martis, based on the morphology of scolex and hooks, with confirmation by PCR amplification and sequence analysis of the mitochondrial cytochrome c oxidase subunit 1 (cox1) and NADH dehydrogenase subunit 1 (nad1) genes. Exeresis of the lesion was not possible and praziquantel (5.7 mg/kg) was given twice at an interval of 3 days. The abdominal mass was greatly diminished upon examination 2 months later and no signs of recurrence were noticed during the following 4 years.
Conclusions
This is the first report of T. martis cysticercosis in a monkey. This record and the recent first description of an ocular T. martis cysticercosis in a human show the susceptibility of primates to T. martis and its zoonotic potential. This taeniid species must be considered in the differential diagnosis of cysticercosis in primates.
doi:10.1186/1756-3305-7-422
PMCID: PMC4167275  PMID: 25189669
Taenia martis; Cysticercosis; Zoonosis; Macaca tonkeana; France
3.  The genomes of four tapeworm species reveal adaptations to parasitism 
Nature  2013;496(7443):57-63.
Summary
Tapeworms cause debilitating neglected diseases that can be deadly and often require surgery due to ineffective drugs. Here we present the first analysis of tapeworm genome sequences using the human-infective species Echinococcus multilocularis, E. granulosus, Taenia solium and the laboratory model Hymenolepis microstoma as examples. The 115-141 megabase genomes offer insights into the evolution of parasitism. Synteny is maintained with distantly related blood flukes but we find extreme losses of genes and pathways ubiquitous in other animals, including 34 homeobox families and several determinants of stem cell fate. Tapeworms have species-specific expansions of non-canonical heat shock proteins and families of known antigens; specialised detoxification pathways, and metabolism finely tuned to rely on nutrients scavenged from their hosts. We identify new potential drug targets, including those on which existing pharmaceuticals may act. The genomes provide a rich resource to underpin the development of urgently needed treatments and control.
doi:10.1038/nature12031
PMCID: PMC3964345  PMID: 23485966
HSP70; parasitism; Cestoda; cysticercosis; echinococcosis; Platyhelminthes
4.  Dynamics of the Force of Infection: Insights from Echinococcus multilocularis Infection in Foxes 
Characterizing the force of infection (FOI) is an essential part of planning cost effective control strategies for zoonotic diseases. Echinococcus multilocularis is the causative agent of alveolar echinococcosis in humans, a serious disease with a high fatality rate and an increasing global spread. Red foxes are high prevalence hosts of E. multilocularis. Through a mathematical modelling approach, using field data collected from in and around the city of Zurich, Switzerland, we find compelling evidence that the FOI is periodic with highly variable amplitude, and, while this amplitude is similar across habitat types, the mean FOI differs markedly between urban and periurban habitats suggesting a considerable risk differential. The FOI, during an annual cycle, ranges from (0.1,0.8) insults (95% CI) in urban habitat in the summer to (9.4, 9.7) (95% CI) in periurban (rural) habitat in winter. Such large temporal and spatial variations in FOI suggest that control strategies are optimal when tailored to local FOI dynamics.
Author Summary
Human alveolar echinococcosis (AE) is caused by the fox tapeworm E. multilocularis and has a high fatality rate if untreated. The frequency of the tapeworm in foxes can be reduced through the regular distribution of anthelmintic baits and thus decrease the risk of zoonotic transmission. Here, we estimate the force of infection to foxes using a mathematical model and data from necropsied foxes. The results suggest that the frequency of anthelmintic baiting of foxes can be optimised to local variations in transmission that depend upon season and type of fox habitat.
doi:10.1371/journal.pntd.0002731
PMCID: PMC3961194  PMID: 24651596
5.  Evaluation of a rapid device for serological in-clinic diagnosis of canine angiostrongylosis 
Parasites & Vectors  2014;7:72.
Background
Angiostrongylus vasorum is a potentially fatal canine nematode. Due to the high variability of clinical signs and the often chronic and subtle course of the infections, the diagnosis is particularly challenging. A rapid in-clinic assay (Angio Detect™ Test, IDEXX Laboratories, Westbrook, Maine, USA) for the serological detection of circulating antigen and intended for routine in-clinic diagnosis has been evaluated.
Methods
Sensitivity was calculated with sera from 39 naturally infected dogs confirmed by Baermann-Wetzel analysis, while sera of 38 experimentally infected dogs were used for follow-up analyses, of which 10 were treated with imidacloprid/moxidectin. Cross-reactivity was tested with a total of 123 samples from dogs with proven parasitic infections with Toxocara canis (n = 21), Ancylostoma caninum (n = 4), Crenosoma vulpis (n = 18), Oslerus osleri (n = 3), Eucoleus aerophilus, (n = 6), Dirofilaria immitis (n = 28), Dirofilaria repens (n = 20), Acantocheilonema reconditum (n = 10) or Dipetalonema dracunculoides (n = 10) or multiple infections (n = 3). All sera were tested with the Angio Detect™ Test and with an ELISA for detection of circulating antigen of A. vasorum.
Results
The sensitivity of the Angio Detect™ Test was 84.6% (95% C.I. 69.5 - 94.1%), while specificity was 100% (95% C.I. 97.6 - 100%). The sensitivity of the ELISA (94.9%, 95% C.I. 82.7 – 99.3%) was comparable with previous evaluations. In experimentally infected dogs, earliest positive results with the Angio Detect™ Test were observed 9 weeks post inoculation and 5 weeks later all sera were Angio Detect™ Test positive. After anthelmintic treatment, seropositive dogs turned negative again within 3 to 7 weeks after treatment. The evaluation of the colour intensity of the test strips confirmed the delay of approximately 3-4 weeks for antigen detection by the Angio Detect™ Test compared to the ELISA and its correlation with the time after infection.
Conclusions
This study provided evidence of a good sensitivity and a very high specificity of the rapid device Angio Detect™ Test for detection of circulating A. vasorum antigen in dogs with suspected canine angiostrongylosis, representing a very simple and useful tool to be broadly applied in veterinary practices. The rapid detection of infected dogs is a key point for initiating an indispensable and urgent therapy.
doi:10.1186/1756-3305-7-72
PMCID: PMC3936995  PMID: 24548539
Angiostrongylus vasorum; Dogs; Serological diagnosis; Rapid device test; Antigen detection; Specificity; Sensitivity
6.  Human Alveolar Echinococcosis in Kyrgyzstan 
Emerging Infectious Diseases  2013;19(7):1095-1097.
Human echinococcosis is a reportable disease in Kyrgyzstan. Between 1995 and 2011, human alveolar echinococcosis increased from <3 cases per year to >60 cases per year. The origins of this epidemic, which started in 2004, may be linked to the socioeconomic changes that followed the dissolution of the former Soviet Union.
doi:10.3201/eid1907.121405
PMCID: PMC3713972  PMID: 23763935
Echinococcus multilocularis; emergence; Alveolar echinococcosis; Kyrgyzstan; Central Asia; tapeworms; parasites; zoonoses
7.  Evaluation of a commercial ELISA kit for detection of antibodies against Toxoplasma gondii in serum, plasma and meat juice from experimentally and naturally infected sheep 
Parasites & Vectors  2013;6:85.
Background
Toxoplasmosis is one of the most common food borne zoonoses worldwide, and can be a serious life-threatening disease in the congenitally infected fetus and in immunosupressed patients. Among food animals, sheep along with goats and pigs possess the highest incidence of T. gondii cysts in meat, and play a major role as a source of human infection.
Methods
In this study, a new commercial ELISA kit (PrioCHECK® Toxoplasma Ab SR, Prionics Schlieren-Zurich, Switzerland) for the detection of anti-T. gondii antibodies in serum, plasma and meat juice of sheep, was evaluated by comparing it with the indirect fluorescent antibody test (IFAT), indirect haemagglutination test (IHA) and real-time PCR, on samples from experimentally inoculated and naturally exposed sheep.
Results
The commercial ELISA detected the infection status in 50% and 100% of sheep orally inoculated with 10,000 T. gondii oocysts (n = 6), from two or three weeks post infection (wpi), respectively, both on serum and plasma samples. Meat juice from all experimentally inoculated sheep collected at slaughter (12 wpi) showed positive ELISA values. In naturally exposed sheep (n = 396), the ELISA showed a very good agreement with IFAT (kappa = 0.91-1.0) and IHA (kappa = 0.96-1.0) performed on serum; and a positive correlation was observed between ELISA values and IFAT titers. By a Receiver Operating Characteristics (ROC) curve analysis, the commercial ELISA had relative sensitivities between 93.33% and 100%, and relative specificities between 96.87% and 100% respect to IFAT and IHA, depending on the considered cut-off value and animal groups tested. Furthermore, the ELISA correctly recognized all animals reacting positive in real-time PCR. The ELISA results on meat juice agreed with those on serum samples in all experimentally inoculated animals, and in 94 out of 96 (97.9%) naturally exposed sheep, when meat juice was tested at a 1:10 dilution.
Conclusion
The commercial ELISA kit evaluated in this study could represent a valuable tool to improve the surveillance and reporting system for T. gondii in sheep populations at the farm level or for diagnosis at the slaughterhouse, contributing to the control of this widespread zoonosis.
doi:10.1186/1756-3305-6-85
PMCID: PMC3631134  PMID: 23561035
Toxoplasma gondii; Sheep; Diagnosis; ELISA; IFAT; Indirect haemagglutination; Antibodies; Meat juice
8.  Morphometric analyses of canine blood microfilariae isolated by the Knott’s test enables Dirofilaria immitis and D. repens species-specific and Acanthocheilonema (syn. Dipetalonema) genus-specific diagnosis 
Parasites & Vectors  2013;6:48.
Background
Considering the increasing importance of small animals travel medicine and the spread of filariae with zoonotic potential to non-endemic European areas, routine filarial diagnosis in dogs is becoming important. Dirofilaria immitis, D. repens, Acanthocheilonema dracunculoides and A. reconditum are the most common canine filarial nematodes presenting blood circulating microfilariae (mf) which can be differentiated to species level by the acid phosphatase activity patterns or by PCR. Available data on the size of the mf vary considerably in the literature. The aim of this study was to validate morphometric criteria for filarial identification in blood samples of dogs after concentration of mf with the modified Knott’s technique.
Methods
Morphometric analysis of 10 mf from samples identified to species level by acid phosphatase activity and partially confirmed by PCR were performed with specimens from 377 dogs.
Results
The mean length and width of D. immitis mf from 60 dogs were 301.77±6.29 μm and 6.30±0.26 μm, of D. repens mf from 171 dogs 369.44±10.76 μm 8.87±0.58 μm, of A. dracunculoides mf from 133 dogs 259.43±6.69 μm and 5.09±0.47 μm and of A. reconditum mf from 13 dogs 264.83±5.47 μm and 4.63±0.52 μm.
For a subset of 30 samples, morphometric analysis was repeated with identical results in two laboratories. Furthermore, the size of mf concentrated and fixed by the Knott’s technique was shown to be stable over 105 days.
Conclusions
The Knott’s test enables to clearly distinguish between D. immitis, D. repens and Acanthocheilonema spp. However, due to the overlapping size ranges of A. dracunculoides and A. reconditum, biochemical or molecular methods are required to distinguish these two species.
doi:10.1186/1756-3305-6-48
PMCID: PMC3598535  PMID: 23442771
Canine blood microfilariae; Laboratory diagnosis; Size of microfilariae
9.  Toxoplasma gondii Infection in Kyrgyzstan: Seroprevalence, Risk Factor Analysis, and Estimate of Congenital and AIDS-Related Toxoplasmosis 
Background
HIV-prevalence, as well as incidence of zoonotic parasitic diseases like cystic echinococcosis, has increased in the Kyrgyz Republic due to fundamental socio-economic changes after the breakdown of the Soviet Union. The possible impact on morbidity and mortality caused by Toxoplasma gondii infection in congenital toxoplasmosis or as an opportunistic infection in the emerging AIDS pandemic has not been reported from Kyrgyzstan.
Methodology/Principal Findings
We screened 1,061 rural and 899 urban people to determine the seroprevalence of T. gondii infection in 2 representative but epidemiologically distinct populations in Kyrgyzstan. The rural population was from a typical agricultural district where sheep husbandry is a major occupation. The urban population was selected in collaboration with several diagnostic laboratories in Bishkek, the largest city in Kyrgyzstan. We designed a questionnaire that was used on all rural subjects so a risk-factor analysis could be undertaken. The samples from the urban population were anonymous and only data with regard to age and gender was available. Estimates of putative cases of congenital and AIDS-related toxoplasmosis in the whole country were made from the results of the serology. Specific antibodies (IgG) against Triton X-100 extracted antigens of T. gondii tachyzoites from in vitro cultures were determined by ELISA. Overall seroprevalence of infection with T. gondii in people living in rural vs. urban areas was 6.2% (95%CI: 4.8–7.8) (adjusted seroprevalence based on census figures 5.1%, 95% CI 3.9–6.5), and 19.0% (95%CI: 16.5–21.7) (adjusted 16.4%, 95% CI 14.1–19.3), respectively, without significant gender-specific differences. The seroprevalence increased with age. Independently low social status increased the risk of Toxoplasma seropositivity while increasing numbers of sheep owned decreased the risk of seropositivity. Water supply, consumption of unpasteurized milk products or undercooked meat, as well as cat ownership, had no significant influence on the risk for seropositivity.
Conclusions
We present a first seroprevalence analysis for human T. gondii infection in the Kyrgyz Republic. Based on these data we estimate that 173 (95% CI 136–216) Kyrgyz children will be born annually to mothers who seroconverted to toxoplasmosis during pregnancy. In addition, between 350 and 1,000 HIV-infected persons are currently estimated to be seropositive for toxoplasmosis. Taken together, this suggests a substantial impact of congenital and AIDS-related symptomatic toxoplasmosis on morbidity and mortality in Kyrgyzstan.
Author Summary
A serological study on toxoplasmosis was undertaken in a rural and urban population in Kyrgyzstan. The observed seroprevalence was adjusted because of differences between age and gender stratifications in the study group compared to population census figures. This gave an estimated seroprevalence in rural and urban populations of 5.1% and 16.4% respectively. In our analysis we determined the risk-factors for infection in the rural population to be age, low social-status and low number of sheep owned. While the seroprevalence in this rural population was relatively low, the seroprevalence found in the urban population of Bishkek correlated better with international data. Extrapolating from our data, about 173 seroconversions during pregnancy may be expected annually in Kyrgyzstan. In addition, considering a prevalence of HIV-Toxoplasma-co-infection between 7/100,000 (official HIV-prevalence data) and 19.4/100,000 (UNAIDS-estimates), 350–1,000 people are at risk for AIDS-related toxoplasmosis. Therefore, in the face of the rising prevalence of HIV infection education of medical personnel on treatment and prevention of toxoplasmosis is recommended.
doi:10.1371/journal.pntd.0002043
PMCID: PMC3566989  PMID: 23409201
10.  A Multiplex PCR for the Simultaneous Detection and Genotyping of the Echinococcus granulosus Complex 
Echinococcus granulosus is characterized by high intra-specific variability (genotypes G1–G10) and according to the new molecular phylogeny of the genus Echinococcus, the E. granulosus complex has been divided into E. granulosus sensu stricto (G1–G3), E. equinus (G4), E. ortleppi (G5), and E. canadensis (G6–G10). The molecular characterization of E. granulosus isolates is fundamental to understand the spatio-temporal epidemiology of this complex in many endemic areas with the simultaneous occurrence of different Echinococcus species and genotypes. To simplify the genotyping of the E. granulosus complex we developed a single-tube multiplex PCR (mPCR) allowing three levels of discrimination: (i) Echinococcus genus, (ii) E. granulosus complex in common, and (iii) the specific genotype within the E. granulosus complex. The methodology was established with known DNA samples of the different strains/genotypes, confirmed on 42 already genotyped samples (Spain: 22 and Bulgaria: 20) and then successfully applied on 153 unknown samples (Tunisia: 114, Algeria: 26 and Argentina: 13). The sensitivity threshold of the mPCR was found to be 5 ng Echinoccoccus DNA in a mixture of up to 1 µg of foreign DNA and the specificity was 100% when template DNA from closely related members of the genus Taenia was used. Additionally to DNA samples, the mPCR can be carried out directly on boiled hydatid fluid or on alkaline-lysed frozen or fixed protoscoleces, thus avoiding classical DNA extractions. However, when using Echinococcus eggs obtained from fecal samples of infected dogs, the sensitivity of the mPCR was low (<40%). Thus, except for copro analysis, the mPCR described here has a high potential for a worldwide application in large-scale molecular epidemiological studies on the Echinococcus genus.
Author Summary
The dog tapeworm Echinococcus granulosus (E. granulosus) is a cosmopolitan parasite. The adult worms reside in the small intestine of their definitive hosts (dogs). Infective eggs are shed with the feces into the environment and are orally ingested by intermediate hosts where they develop into the metacestode (larval) stage, causing cystic echinococcosis (CE) in humans and livestock. Ten intraspecific genotypes of E. granulosus (G1 to G10) have been reported from different intermediate host species. Based on the recently established molecular phylogeny, E. granulosus is now considered a complex consisting of four species: E. granulosus sensu stricto (G1/G2/G3), E. equinus (G4), E. ortleppi (G5) and E. canadensis (G6–G10). Simple and highly discriminative molecular epidemiological approaches are needed to explore dynamics, life cycle patterns, and the pathogenicity of the members of this complex. We here introduce a one-step multiplex PCR (mPCR) protocol for the genotyping and discrimination of the different members of the E. granulosus complex, allowing three levels of discrimination: (i) Echinococcus genus, (ii) E. granulosus complex, and (iii) genetic variants within the E. granulosus complex. The relatively complicated task of E. granulosus complex speciation and genotyping is clearly simplified by mPCR, and this technique therefore represents a useful tool for routine practice.
doi:10.1371/journal.pntd.0002017
PMCID: PMC3547860  PMID: 23350011
11.  Taeniid species of the Iberian wolf (Canis lupus signatus) in Portugal with special focus on Echinococcus spp.☆ 
Graphical abstract
Highlights
► This is the first study on the taeniid species infecting the Portuguese population of Iberian wolf. ► Taeniid species detected are strongly related to the feeding habits of this top carnivore. ► First report of Taenia polyacantha and Echinococcus intermedius in the Iberian wolf. ► A genuine wild cycle for E. intermedius might persist between wolves and wild boars in this region.
Taeniid species represent relevant pathogens in human and animals, circulating between carnivorous definitive hosts and a variety of mammalian intermediate hosts. In Portugal, however, little is known about their occurrence and life cycles, especially in wild hosts. An epidemiological survey was conducted to clarify the role of the Iberian wolf as a definitive host for taeniid species, including Echinococcus spp. Wolf fecal samples (n = 68) were collected from two regions in Northern Portugal. Taeniid eggs were isolated through a sieving-flotation technique, and species identification was performed using multiplex-PCR followed by sequencing of the amplicons. Taenia hydatigena (in 11.8% of the samples), Taenia serialis (5.9%), Taenia pisiformis (2.9%), Taenia polyacantha (1.5%) and Echinococcus intermedius (Echinococcus granulosus ‘pig strain’, G7) (1.5%) were detected. This is the first study to characterize the taeniid species infecting the Portuguese Iberian wolf, with the first records of T. polyacantha and E. intermedius in this species in the Iberian Peninsula. Iberian wolves can be regarded as relevant hosts for the maintenance of the wild and synanthropic cycles of taeniids in Portugal.
doi:10.1016/j.ijppaw.2012.11.007
PMCID: PMC3862541  PMID: 24533315
Echinococcus intermedius; Taenia spp.; Iberian wolf; Portugal
12.  Cross-reactions of sera from dogs infected with Angiostrongylus vasorum in commercially available Dirofilaria immitis test kits 
Parasites & Vectors  2012;5:258.
Background
Dirofilaria immitis and Angiostrongylus vasorum are both important potentially fatal canine nematodes with overlapping endemic areas, especially in Europe. The preadult and adult stages of both species are living in the Arteria pulmonalis and the right heart, and diagnostically detectable circulating parasite antigens have been demonstrated for both species. For the detection of D. immitis infections, a variety of commercial tests have been developed, however, they have not been evaluated for cross-reactions against circulating antigens of A. vasorum.
Methods
In this study, potential cross-reactions of sera from 16 dogs, which were experimentally infected with A. vasorum and which had circulating antigens as confirmed by a species-specific ELISA, were evaluated for the detection of A. vasorum antigen in six commercially available D. immitis test kits.
Results
In three fast tests (Witness® Dirofilaria, SensPERT® Canine Heartworm, SNAP® 4Dx® Plus), all sera were negative. One fast membrane ELISA (SNAP® HTWM RT Test) was positive with four sera (25%), and one serum delivered a non-valid result twice. In the PetChek® HTWM PF Test, depending on the interpretation protocol, 5 or 8 dogs (31.2 – 50%) were positive. With the DiroCHEK®-ELISA, a single A. vasorum-infected dog (6.2%) tested positive.
Conclusions
Due to potential cross-reactions with A. vasorum in commercially available test kits for the detection of D. immitis antigen, the simultaneous use of highly specific diagnostic methods for the differentiation of these two canine heart worms is recommended.
doi:10.1186/1756-3305-5-258
PMCID: PMC3503614  PMID: 23148786
Angiostrongylus vasorum; Dirofilaria immitis; Antigen detection; Cross-reactions; Dogs
13.  Sensitive and Specific Immunohistochemical Diagnosis of Human Alveolar Echinococcosis with the Monoclonal Antibody Em2G11 
Background
Alveolar echinococcosis (AE) is caused by the metacestode stage of Echinococcus multilocularis. Differential diagnosis with cystic echinococcosis (CE) caused by E. granulosus and AE is challenging. We aimed at improving diagnosis of AE on paraffin sections of infected human tissue by immunohistochemical testing of a specific antibody.
Methodology/Principal Findings
We have analysed 96 paraffin archived specimens, including 6 cutting needle biopsies and 3 fine needle aspirates, from patients with suspected AE or CE with the monoclonal antibody (mAb) Em2G11 specific for the Em2 antigen of E. multilocularis metacestodes. In human tissue, staining with mAb Em2G11 is highly specific for E. multilocularis metacestodes while no staining is detected in CE lesions. In addition, the antibody detects small particles of E. multilocularis (spems) of less than 1 µm outside the main lesion in necrotic tissue, liver sinusoids and lymphatic tissue most probably caused by shedding of parasitic material. The conventional histological diagnosis based on haematoxylin and eosin and PAS stainings were in accordance with the immunohistological diagnosis using mAb Em2G11 in 90 of 96 samples. In 6 samples conventional subtype diagnosis of echinococcosis had to be adjusted when revised by immunohistology with mAb Em2G11.
Conclusions/Significance
Immunohistochemistry with the mAb Em2G11 is a new, highly specific and sensitive diagnostic tool for AE. The staining of small particles of E. multilocularis (spems) outside the main lesion including immunocompetent tissue, such as lymph nodes, suggests a systemic effect on the host.
Author Summary
Echinococcosis is a life-threatening disease in humans that is caused by the larval stages of the tapeworms Echinococcus multilocularis and Echinococcus granulosus. The eggs of the parasites are released with faeces of canids, and humans are aberrantly infected. In humans, the larval stages of the parasites cause tumour-like lesions mainly in the liver and the lungs. Precise diagnosis of the parasite responsible for human disease is of utmost importance since therapy regimens largely differ between cystic and alveolar echinococcosis. Diagnosis is based on serology, imaging and histology, the latter being the gold standard. However, conventional histology cannot always clearly identify the causative parasite because both parasites can cause human tissue to present similar features. Therefore, we have developed the monoclonal antibody Em2G11 and an immunohistological technique that allows a cheap and fast clear-cut diagnosis of E. multilocularis even on aspirates and small archived bioptic tissue samples. Furthermore, this technique disclosed an unknown feature of human alveolar echinococosis we called "small particles of E. multilocularis" (spems). We argue that these small particles represent micro-fragments of E. multilocularis and thus point to a new form of host-parasite interaction.
doi:10.1371/journal.pntd.0001877
PMCID: PMC3493387  PMID: 23145198
14.  Age, season and spatio-temporal factors affecting the prevalence of Echinococcus multilocularis and Taenia taeniaeformis in Arvicola terrestris 
Background
Taenia taeniaeformis and the related zoonotic cestode Echinococcus multilocularis both infect the water vole Arvicola terrestris. We investigated the effect of age, spatio-temporal and season-related factors on the prevalence of these parasites in their shared intermediate host. The absolute age of the voles was calculated based on their eye lens weights, and we included the mean day temperature and mean precipitation experienced by each individual as independent factors.
Results
Overall prevalences of E. multilocularis and T. taeniaeformis were 15.1% and 23.4%, respectively, in 856 A. terrestris trapped in the canton Zürich, Switzerland. Prevalences were lower in young (≤ 3 months: E. multilocularis 7.6%, T. taeniaeformis 17.9%) than in older animals (>7 months: 32.6% and 34.8%). Only 12 of 129 E. multilocularis-infected voles harboured protoscoleces. Similar proportions of animals with several strobilocerci were found in T. taeniaeformis infected voles of <5 months and ≥5 months of age (12.8% and 11.9%). Multivariate analyses revealed strong spatio-temporal variations in prevalences of E. multilocularis. In one trapping area, prevalences varied on an exceptional high level of 40.6-78.5% during the whole study period. Low temperatures significantly correlated with the infection rate whereas precipitation was of lower importance. Significant spatial variations in prevalences were also identified for Taenia taeniaeformis. Although the trapping period and the meteorological factors temperature and precipitation were included in the best models for explaining the infection risk, their effects were not significant for this parasite.
Conclusions
Our results demonstrate that, besides temporal and spatial factors, low temperatures contribute to the risk of infection with E. multilocularis. This suggests that the enhanced survival of E. multilocularis eggs under cold weather conditions determines the level of infection pressure on the intermediate hosts and possibly also the infection risk for human alveolar echincoccosis (AE). Therefore, interventions against the zoonotic cestode E. multilocularis by deworming foxes may be most efficient if conducted just before and during winter.
doi:10.1186/1756-3305-4-6
PMCID: PMC3033848  PMID: 21247427
18.  Molecular Characterization of EmABP, an Apolipoprotein A-I Binding Protein Secreted by the Echinococcus multilocularis Metacestode▿  
Infection and Immunity  2009;77(12):5564-5571.
Cestodes are unable to synthesize de novo most of their own membrane lipids, including cholesterol, and have to take them up from the host during an infection. The underlying molecular mechanisms are so far unknown. Here we report the identification and characterization of a novel gene, Emabp, which is expressed by larval stages and adults of the fox tapeworm Echinococcus multilocularis. The encoded protein, EmABP, displays significant homologies to apolipoprotein A-I binding protein (AI-BP) of mammalian origin and to metazoan YjeF_N domain proteins. Like mammalian AI-BP, EmABP carries an export-directing signal sequence which is absent in predicted AI-BP orthologs from the related flatworms Schistosoma japonicum and Schmidtea mediterranea. Using a specific antibody and immunoprecipitation techniques, we demonstrate that EmABP is secreted into the extraparasitic environment and into the hydatid fluid of in vitro-cultivated metacestode vesicles. Furthermore, we show that apolipoprotein A-I (apoA-I), a major constituent of cholesterol-transporting high-density lipoproteins, is present in hydatid fluid. By pulldown experiments, we demonstrate that recombinantly expressed, purified EmABP interacts with purified human apoA-I and is able to precipitate apoA-I from human serum. On the basis of these features and the suggested function of AI-BP in cholesterol transport in higher eukaryotes, we propose a role for EmABP in cholesterol and lipid uptake mechanisms of larval E. multilocularis.
doi:10.1128/IAI.00653-09
PMCID: PMC2786436  PMID: 19805524
19.  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.
Author Summary
Echinococcus multilocularis is a tapeworm of the red fox, which represents a considerable health threat to respectively infected humans. Main endemic areas are located in China, Siberia, and central Europe. Alarmed by an emerging or reemerging situation in Europe, the question of how the parasite gets spatially and temporally spread and transmitted becomes essential to prepare appropriate control programs. The question was tackled by using genetic data on a large sample size of E. multilocularis adult stage tapeworms, combined with geographical site location data input. The historically documented endemic area, represented by the northern Alpine arch, was shown to harbour the highest genetic richness and diversity, as compared to surrounding areas in northern and eastern Europe. The spatial and temporal spread of different E. multilocularis genotypes in Europe seems to be ruled by a founder event, linked to exportation of parasites from the central core to newly identified (western and eastern) areas or subregions, where these parasites could subsequently disseminate under geographical separation from the original foci.
doi:10.1371/journal.pntd.0000452
PMCID: PMC2685985  PMID: 19513103
20.  Control Strategy for Echinococcus multilocularis 
Emerging Infectious Diseases  2008;14(10):1626-1628.
Echinococcus multilocularis, the causative agent of zoonotic alveolar echinococcosis, can be controlled effectively by the experimental delivery of anthelminthic baits for urban foxes. Monthly baiting over a 45-month period was effective for long-lasting control. Trimonthly baiting intervals were far less effective and did not prevent parasite recovery.
doi:10.3201/eid1410.080522
PMCID: PMC2609876  PMID: 18826831
Control; Echinococcus multilocularis; praziquantel; urbanization; vulpes; dispatch
21.  Survey of public knowledge about Echinococcus multilocularis in four European countries: Need for proactive information 
BMC Public Health  2008;8:247.
Background
Public information about prevention of zoonoses should be based on the perceived problem by the public and should be adapted to regional circumstances. Growing fox populations have led to increasing concern about human alveolar echinococcosis, which is caused by the fox tapeworm Echinococcus multilocularis. In order to plan information campaigns, public knowledge about this zoonotic tapeworm was assessed.
Methods
By means of representative telephone interviews (N = 2041), a survey of public knowledge about the risk and the prevention of alveolar echinococcosis was carried out in the Czech Republic, France, Germany and Switzerland in 2004.
Results
For all five questions, significant country-specific differences were found. Fewer people had heard of E. multilocularis in the Czech Republic (14%) and France (18%) compared to Germany (63%) and Switzerland (70%). The same effect has been observed when only high endemic regions were considered (Czech Republic: 20%, France: 17%, Germany: 77%, Switzerland: 61%). In France 17% of people who knew the parasite felt themselves reasonably informed. In the other countries, the majority felt themselves reasonably informed (54–60%). The percentage that perceived E. multilocularis as a high risk ranged from 12% (Switzerland) to 43% (France). In some countries promising measures as deworming dogs (Czech Republic, Switzerland) were not recognized as prevention options.
Conclusion
Our results and the actual epidemiological circumstances of AE call for proactive information programs. This communication should enable the public to achieve realistic risk perception, give clear information on how people can minimize their infection risk, and prevent exaggerated reactions and anxiety.
doi:10.1186/1471-2458-8-247
PMCID: PMC2522376  PMID: 18644138
22.  Spatial Distribution of Echinococcus multilocularis, Svalbard, Norway 
Emerging Infectious Diseases  2008;14(1):73-75.
In Svalbard, Norway, the only intermediate host for Echinococcus multilocularis, the sibling vole, has restricted spatial distribution. A survey of feces from the main host, the arctic fox, showed that only the area occupied by the intermediate host is associated with increased risk for human infection.
doi:10.3201/eid1401.070565
PMCID: PMC2600161  PMID: 18258082
Vulpes lagopus; arctic fox; Microtus levis; sibling vole; Echinococcus multilocularis; ELISA; coproantigen; dispatch
23.  Alveolar Echinococcosis, Lithuania 
Emerging Infectious Diseases  2007;13(10):1618-1619.
Alveolar Echniococcosis, Lithuania
doi:10.3201/eid1310.061161
PMCID: PMC2851530  PMID: 18258025
Echinococcosis; hepatic alveolar; Echinococcus multilocularis; fox; Lithuania; letter
24.  Human Alveolar Echinococcosis after Fox Population Increase, Switzerland 
Emerging Infectious Diseases  2007;13(6):878-882.
An increase in fox population has led to an increase in incidence of human alveolar echinococcosis.
We analyzed databases spanning 50 years, which included retrospective alveolar echinococcosis (AE) case-finding studies and databases of the 3 major centers for treatment of AE in Switzerland. A total of 494 cases were recorded. Annual incidence of AE per 100,000 population increased from 0.12– 0.15 during 1956–1992 and a mean of 0.10 during 1993–2000 to a mean of 0.26 during 2001–2005. Because the clinical stage of the disease did not change between observation periods, this increase cannot be explained by improved diagnosis. Swiss hunting statistics suggested that the fox population increased 4-fold from 1980 through 1995 and has persisted at these higher levels. Because the period between infection and development of clinical disease is long, the increase in the fox population and high Echinococcus multilocularis prevalence rates in foxes in rural and urban areas may have resulted in an emerging epidemic of AE 10–15 years later.
doi:10.3201/eid1306.061074
PMCID: PMC2792858  PMID: 17553227
Alveolar echinococcosis; Echinococcus multilocularis; epidemiology; fox (Vulpes vulpes); zoonosis; incidence; Switzerland; research
25.  Babesia spp. Identified by PCR in Ticks Collected from Domestic and Wild Ruminants in Southern Switzerland 
Applied and Environmental Microbiology  2006;72(10):6503-6507.
Concurrent infections with vector-borne pathogens affected a cattle herd in Switzerland, and one of the pathogens was identified as Babesia bigemina, which had never been observed in this country before. Therefore, a survey of the occurrence of ruminant Babesia spp. and their tick vectors in Switzerland was conducted. A total of 2,017 ticks were collected from sheep, goats, cattle, and wild ruminants (deer, roe deer, and chamois) in southern parts of Switzerland and identified morphologically. The vast majority of the ticks (99.2%) were Ixodes ricinus, but 14 ticks from sheep and goats were identified as Dermacentor marginatus and two ticks from wild ruminants were identified as Hemaphysalis punctata. PCR analyses of 700 ticks revealed the presence of Babesia divergens (n = 6), Babesia sp. genotype EU1 (n = 14), and B. major (n = 2), whose suggested occurrence was confirmed in this study by molecular analysis, and the presence of novel Babesia sp. genotype CH1 (n = 4), which is closely related to B. odocoilei and to Babesia sp. genotype RD61 reported from North America. The identification of B. divergens and B. major in ticks collected from wild ruminants cast doubt on the postulated strict host specificity of these bovine Babesia species. Furthermore, the zoonotic Babesia sp. genotype EU1 was detected in ticks collected from domestic animals but was obtained predominantly from ticks collected from wild ruminants. More than one tick containing DNA of different Babesia spp. were collected from two red deer. Hence, the role of these game animals as reservoir hosts of Babesia spp. seems to be important but requires further investigation.
doi:10.1128/AEM.00823-06
PMCID: PMC1610307  PMID: 17021198

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