Control Tools

• Diagnostics availability

• Commercial diagnostic kits available worldwide

  Yes for human cystic echinococcosis (Echinococcus granulosus s.l.) and alveolar echinococcosis (E. multilocularis) serum antibody testing. No commercial tests available for animals.

  GAP:

  Most based on native crude or purified antigens, few recombinant antigens (generally, sensitivity can strongly vary in endemic areas and is dependent on the stage of infection, small developing cysts and degenerated cysts are often not detected serologically).

• Commercial diagnostic kits available in Europe

  Human cystic echinococcosis and alveolar echinococcosis serum antibody testing (haemagglutination tests, IFAT, ELISA, WB)Diagnostic genetic identification (DNA amplification by PCR and sequencing) using clinical material as samples (faeces of definitive hosts, biopsy or necropsy material from intermediate and dead-end hosts) routinely performed in diagnostic laboratories (specific primers published are free available)

  GAP:

  No commercial coproantigen ELISAs for E. granulosus s.l. or E. multilocularis in dogs or foxes is available.Some commercial tests available in China with not well documented diagnostic values.Molecular identification in specialised laboratories, some of the approaches for the detection of copro DNA are time consuming and expensive.

• Diagnostic kits validated by International, European or National Standards

  No

• Diagnostic method(s) described by International, European or National standards

  Routine methods are described in the OIE Manual of Diagnostic Tests and Vaccines.

  Identification of the agent:

  In the intermediate host, diagnosis depends on the detection of the larval cyst form, which can occur in almost any organ, but particularly in the liver and lungs.

  Ultrasonography has been used with good SP and SE values, even if it is time consuming and requires experienced technicians. Actually, it can be performed only in the liver.

  In the definitive host the diagnosis of Echinococcus spp. requires the demonstration of the adult cestodes of Echinococcus spp. in the small intestine or the detection of genus-specific coproantigens or parasite’-DNA in faeces. Copro-DNA and especially egg DNA has proven to be of value for the diagnosis of intestinal Echinococcus spp. DNA and taeniid egg isolation from the faeces is feasible, however, is laborious and expensive and laboratory DNA contamination has to be avoided.

  Serological testsa) Intermediate hostsImmunological tests, useful in humans, are less sensitive and specific in livestock and at present cannot replace necropsy or meet inspection.b) Definitive hostsThis approach is presently of limited practical use.

  GAP:

  The application of Echino CoproELISAs have mainly been replaced by DNA detection (no commercial ELISA test available in EU market). However, studies on validation of commercial DNA extraction kits showed, that significant differences in removal of PCR inhibitors are present between the tested kits. These results suggest that each commercial DNA extraction kit should be validated in the lab with reference faecal samples before routine use. It was also shown, that none of the DNA extraction kits tested was able to remove PCR inhibitors completely. Significant differences in the performance of different PCR protocols in the presence of coextracted inhibitors were recorded, suggesting that these have also to be validated before routine use. (e.g. https://www.ncbi.nlm.nih.gov/pubmed/28268038).

  No sufficiently specific and sensitive serological test for Cystic Echinococcosis (CE) in livestock.Serological test for AE in translocated wild/captive mammals to prevent parasite introduction in some species available (primates, pigs).

• Commercial potential for diagnostic kits in Europe

  High potential for the use in individual dogs (coproantigen fast tests) in endemic areas and for conducting surveys in dogs or foxes in Europe and other countries.

  GAP:

  Copro-ELISAs for E. multilocularis or E. granulosus in dogs at risk in endemic areas or to monitor dogs in PET schemes or transboundary movements.

• DIVA tests required and/or available

  Not required at present although if a recombinant vaccines is developed differentiating tests could be developed in tandem

• Opportunities for new developments

  Sensible, informed use of serology (with a high positive predictive value) should be encouraged.Its sole use in surveillance was discouraged by the WHO consultation in June 2011, as imaging findings are the primary source for determining the prevalence and incidence of cystic echinococcosis.

• Vaccines availability

• Commercial vaccines availability (globally)

  Two commercial sources of a livestock vaccine for prevention of infection are available: (1) Manufactured in China and currently being used extensively in a Chinese national echinococcosis control campaign; (2) manufactured in Argentina and currently in use in parts of Argentina and Chile. Vaccine is available from both of these suppliers.

  GAP :

  Registration of one of these products, or a similar product, in other countries which are endemic for cystic echinococcosis. There is a need for application of the vaccine in parts of Europe where the disease persists and, in some areas (e.g. Sardinia), is highly prevalent.

• Commercial vaccines authorised in Europe

  No

  GAP :

  Required

• Marker vaccines available worldwide

  No

  GAP :

  Required

• Marker vaccines authorised in Europe

  No

• Effectiveness of vaccines / Main shortcomings of current vaccines

  Lack of registration of the available products in many endemic countries.

  GAP:

  Needs pilot trials in European CE endemic regions and compare with praziquantel dosing; surveillance required.

• Commercial potential for vaccines in Europe

  There might be several strategies in Europe, including ‘do nothing’ depending on the epidemiological situation. However there is a potential for a sheep vaccine in large parts of South Europe.

  It would be expensive to vaccinate lambs to prevent them becoming infected. Especially in some highly endemic areas (e.g. Sardinia, parts of mainland Italy, Bulgaria, Spain) where other strategies have failed in the past. It is unlikely that the farmers will use it unless the vaccine is incorporated free or at very low cost or mixed with other commercial livestock vaccines. Continuous vaccination of lambs over 5-7 years could be feasible, Farmers could become interested on an Echinococcus vaccine as experienced with a T. multiceps vaccine (point 2.8), in contrast to CE, coenurosis is responsible of important economic losses in the endemic regions.

  There may be more practical advantages in developing a vaccine for dogs to prevent; however there are few experimental studies and none with significant dog numbers and post- patency assessment. Oral vaccines for foxes to prevent infection with E. multilocularis could be advantageous, but little evidence of intestinal immunity in foxes has been documented.

  GAP:

  Scientific basis for the development of vaccines against E. multilocularis and E. granulosus for foxes and dogs is missing. Furthermore there is no epidemiological evidence that protective immunity develops against intestinal Echinococcus infections.

• Regulatory and/or policy challenges to approval

  There are numerous registered vaccines for non-living, recombinant antigen vaccines including in Europe.

  GAP :

  Non-living, recombinant antigen vaccines are not considered GMOs.

• Commercial feasibility (e.g manufacturing)

  Feasible if the demand exists.

• Opportunity for barrier protection

  Vaccine could be used to prevent the spread of infection in dogs and foxes.

  GAP:

  Use of praziquantel baits or vaccine baits for border/island control of E. multilocularis spread.

• Opportunity for new developments

  The EG95 vaccine has been evaluated in field trials in Argentina, China, but, because of their limitations, these trials will not provide clear scientific evidence of the value of EG95 vaccination for the control of cystic echinococcosis. The principal limitations are the accuracy of the methods for measuring disease prevalence and intensity before control activities and for evaluating disease transmission during and after interventions.

  GAP:

  Field trial could be performed with EG95 in areas like Sardinia where animals could be observed under natural conditions for several years. To date local governments and other institutions in the highly endemic areas did not prioritise such control measurements do economic or priority issues.

• Pharmaceutical availability

• Current therapy (curative and preventive)

  Treatment of dogs/foxes at 4-6 weeks intervals or adapted to the endemic situation with Praziquantel free or in a bait form can prevent patent infections, but long-term treatment over years has to be considered.

  GAP:

  Slow release praziquantel for single dog treatment to last up to 1 year would be beneficial.

• Future therapy

  Improved and long lasting products for the definitive hosts.Treatment of intermediate hosts is not practical.

  GAP:

  Improvement of protoscolicidal treatment in intermediate hosts would be of major interest to decrease parasite transmission and mass treatment would be an option at beginning of vaccine interventions.

• Commercial potential for pharmaceuticals in Europe

  Depends on demand and cost, protoscolicidal treatments would have a high commercial potential if control programs financed by the governments.For the farmers a treatment against CE in sheep is not associated with economic advantages and therefore is not a priority.

  GAP:

  New formulations with high efficiency for single applications would be required.

• Regulatory and/or policy challenges to approval

  No specific problems but residuals in meat have to be considered

• Commercial feasibility (e.g manufacturing)

  Feasible provided a policy is introduced for treatment thus developing a market

• Opportunities for new developments

  For intestinal infections, praziquantel is the most used and the most efficient drug. New drugs are urgently needed to be prepared, if resistances would occur.

  Benzimidazoles have over long time a good effect against CE and a parasitostatic effect against Alveolar Echinoccosis (AE). In intermediate hosts of CE long time treatment over month is not feasible. New chemotherapeutic strategies and new formulations are needed to overcome this problem.

• New developments for diagnostic tests

• Requirements for diagnostics development

  1) CE and AE in humans: serology for specific antibody detection has been established with many tests. The tests for detection of circulating antigens are currently not in use.

  2) Definitive hosts: fast tests (molecular tests) for copro-DNA or coproantigens.

  3) Intermediate hosts: serological tests so far not of practical use.

  GAP :

  1) high variability in sensitivity and specificity, often, especially low sensitivity for early detection, tests are not validated. Basic research on secretome of cysts is needed to identify potential circulating metacestode antigens .

  2) lack in specificity of coproantigen detection, expensive copro-DNA isolation, more specific monoclonal antibodies needed.

  3) lack of sensitivity and specificity of antibody detection, specific antigen detection should be developed. Such tests could be used especially at beginning of control programs for CE.

• Time to develop new or improved diagnostics

  From development through validation to commercial availability will be time consuming and can take years.

  GAP:

  Secreted or shed parasite molecules are highly glycosylated, mucin type proteins and therefore difficult to handle, purify and to produce.

• Cost of developing new or improved diagnostics and their validation

  The development and validation of new tests is time consuming and labour intensive which is costly. Costs cannot be specified as they will depend on the nature of the test and the cost of producing reagents and supplying reading or processing machines if necessary. Once validated a commercial company willing to market the test is needed.

  GAP :

  New basic research on proteomics and glycomics of the secretome are needed to identify new diagnostic targets.

• Research requirements for new or improved diagnostics

  Especially secretome analyses of metacestodes would improve the selection of new antigen targets

  GAP :

  Funds for basic research needed.

• Technology to determine virus freedom in animals

  This would be very difficult in the case of echinococcosis as the parasite can persist over years in the intermediate hosts.

• New developments for vaccines

• Requirements for vaccines development / main characteristics for improved vaccines

  The trials with EG95 vaccine indicate that it is highly effective in sheep and that vaccinated lambs do not become infected with E. granulosus. Vaccination therefore interrupts the cycle. Farmers are, however, reluctant to vaccinate sheep when there is no apparent improvement in animal health and also because of the complex logistics of vaccination. The vaccine might also be blamed for losses due to concomitant disease.

  GAP:

  Scientifically rigorous evaluation is needed of potential new regimes for the control of cystic echinococcosis incorporating livestock vaccination. The results of such evaluations would form the basis for a clear, evidence-based plan for future control activities and attract renewed investment in cystic echinococcosis control. Development of new, combination vaccines including EG95 in other commercially valuable livestock vaccines. It is still unclear if the current vaccine is effective against other species or genotypes for example E. intermedius G6/7 which can affect humans.

• Time to develop new or improved vaccines

  Depending on when a candidate vaccine could be identified the timescale will be 5-10 years. This will involve development, clinical trials and licensing. Potential vaccines need to be identified and subjected to initial trials and depending on the outcome will depend the time to commercial availability.

• Cost of developing new or improved vaccines and their validation

  Expensive with the need to develop and undertake all the relevant tests to provide data to enable the product to be authorised. Field trial will be difficult as will evaluating the results.

  GAP :

  Especially dog and fox echinococcus vaccines.

• Research requirements for new or improved vaccines

  None specifiedEG95 is an excellent vaccine for E.granulosus but requires commercial or government backed mass production.

• New developments for pharmaceuticals

• Requirements for pharmaceuticals development

  None specified

• Time to develop new or improved pharmaceuticals

  Time to develop would depend on the product and the trials necessary to validate the efficacy and safety. Commercial production would then take further time. Five to 10 years seems a realistic timeframe

• Cost of developing new or improved pharmaceuticals and their validation

  Expensive but difficult to assess as it will depend on the product and the trials necessary to validate and license.

• Research requirements for new or improved pharmaceuticals

  Pharmaceutics insuring the elimination of the parasite in intermediate hosts are not available. So far no resistances have been documented against praziquantel, the only drug used against intestinal stages.

  GAP :

  New or modified compounds efficient against protoscoleces in single applications would be beneficial for the reduction of the parasite reservoir. Tests to document praziquantel resistance have not been developed.

Disease details

• Description and characteristics

• Pathogen

  In Europe a group of Echinococcus spp., partially subdivided in strains or genotypes have been documented (see Section “Species involved”). A number of intraspecific variants have been described for E. granulosus s.l., which exhibit morphological, biological and genetic characteristics. Some of the E. granulosus s.l. genotypes have been recommended for elevation to species status. Up to 2018, in Europe five genotypes of E. granulosus s.l. (G1-3, 5, 7, 8 and 10) causing cystic echinococcosis in humans have been identified. E. granulosus s.s. G1-3 is most prominently in South and Southeast Europe, E. intermedius G7 is the dominant species in the Baltic states, Poland and bordering countries. E. ortleppi G5, sporadically found in Central Europe and E. canadensis (G8, 10) in Northern Scandinavia and has a zoonotic potential but it has very rarely been identified in humans in recent years in Europe.

  E. multilocularis (European haplotypes) occurs across Central and Eastern Europe.

  GAP:

  The taxonomy is still controversial; however, the genotype nomenclature for species causing CE (G1-10) is well established and accepted and can be expanded for new genotypes.

  Require acceptance of elevating genotypes of E. granulosus s.l. to species level (e.g. E. intermedius for G6 and 7, E. canadensis G 8 and 10.

  For E. multilocularis so far no indications of an association of genetic diversity with pathogenicity.

• Variability of the disease

  The pathogenicity of E. granulosus s.l. is determined by the localisation of the cysts, the expansive character of cyst development, immunity of the hosts and many other parameters. The genotypes G1-3, 5, and 7 are mostly localised in the liver or lungs causing the typical CE, G8 and 10 are mostly detected in the lungs and in most of the cases not causing severe symptoms (G 6 is not prevalent in Europe).Considerable biological variations in the host range is observed, E. granulosus s.s. (G1-3) has mainly sheep as intermediate hosts (only around 10% of the cysts in cattle are fertile), G7 nearly exclusively in pigs and G8 and 10 is circulating between wolfs and cervids.

  GAP:

  Not all human cysts have been genotyped but there is convincing evidence of E. granulosus s.s. and E. intermedius being the most important species causing CE in Europe, for details see section “Pathogens”)

• Stability of the agent/pathogen in the environment

  The eggs are well adapted to survive in the environment for as long as a year in cool moist conditions even under sub-zero temperatures, but are susceptible to desiccation. Fresh eggs are sticky and may adhere to the fur of definitive hosts increasing zoonotic risk.

  GAP :

  Work with viable Echinococcus eggs need high standards of safety. Eggs can only be acquired from in animal experimentally infected or by isolation from freshly hunted wild carnivores. Both methods are costly and require special infrastructure and well trained scientists.

• Species involved

• Animal infected/carrier/disease

  (After Romig et al., 2017):

  Echinococcus spp., strains and genotypes Definitive host (DH) Major intermediate host (IH) E granulosus s.s., sheep strain, G1-3*

  dog and other canids (wolves)

  under natural conditions: sheep, goat, (cattle), E. ortleppi, cattle strain, G5* dog cattle E. intermedius**, pig strain, G7* dog, wolf Pig, wild boar, goats E. canadensis, cervids strains, G8,G10* wolf, dog cervids E. multilocularis fox, raccoon dog, and to a lesser extent dog, wolves, cat

  small mammals esp. vole-like rodents,

  E. equinus* dog equines

  *Species of the zoonotic E. granulosus s.l. complex** proposed species, separated from E. canadensis

  GAP:

  Other species:

  E. equinus, sporadically in Europe, DH: dogs, IH equines (so far not considered to be zoonotic, but recently a case in a human in Asia has been documented).

• Human infected/disease

  Cystic echinococcosis (CE) (old terms: hydatidosis, or hydatid disease) is caused by the larval stages of E. granulosus s.l., mostly in liver or lungs.

  Alveolar echinococcosis (AE) caused by the larval stage of E. multilocularis (mostly in the liver).

  GAP:

  Many reporting systems are not differentiating between CE and AE.

  Early infections and atypical infections are difficult to differentiate and often genetic identification or immunhistological methods are needed.

• Vector cyclical/non-cyclical

  None

• Reservoir (animal, environment)

  Various definitive and intermediate domestic and wild hosts.Eggs can survive for months in the environment.

• Description of infection & disease in natural hosts

• Transmissibility

  Definitive hosts excrete fully embryonated eggs containing a larva (oncosphere). Intermediate hosts (IH) or humans become infected by ingesting eggs with resulting release of oncospheres in the intestine. After invasion through the mucosa the oncospheres reach the bloodstream, developing cysts or cystic lesions in various organs (mainly liver for AE, liver and lungs CE).

  GAP :

  For IH, transmission is linked to the contaminated environment and food. Transmission to humans has been linked to foodborne sources (vegetables/fruits/berries) or to hand-to-mouth transmission, after contact with Echinococcus eggs in the environment (but any source attribution for humans is uncertain due to the long incubation time).

• Pathogenic life cycle stages

  Species involved for disease in humans see Section “Animal infected/carrier/disease”The adult Echinococcus stages reside in the small intestine of the definitive hosts without causing clinical signs.

  The metacestode is causing CE and AE in intermediate or dead–end hosts like humans. Echinococcosis is characterised by a chronic infections with long incubation time (see 10.4).

  GAP:

  Mechanisms of immunopathology and immunomodulation are poorly studied.

• Signs/Morbidity

  There are no signs of disease in definitive hosts.Generally very low morbidity in farm animals as intermediate hosts.

• Incubation period

  Can vary between several month and several years in humans or IH depending on localisation of the metacestode, the species and other factors.

  GAP :

  Many open questions concerning Echinococcus strain or genotype variations, resistance of the host (including immunosuppression, age, risk factors).

• Mortality

  CE: Generally low mortality in IH.AE: high in IH hosts (rodents) with AE

  In humans mortality in untreated cases ranges from <1% to >50%.

  GAP:

  Human AE had a very high mortality (>50%) in untreated cases with hepatic lesions. Treatment of AE needs still improvement in some European countries to reach higher survival rates of patients.

• Shedding kinetic patterns

  After ingestion of metacestodes by a definitive host, the protoscoleces evaginate, attach to the intestinal mucosa, and develop into adult gravid stages in 25 to 80 days (depending on Echinococcus species). The excretion period (patency) is short; the majority of the worm burden is eliminated within 1-3 month of patency, some worms can persist longer with a relative low egg production.

  GAP :

  Patterns of egg production and shedding of E. multilocularis (foxes, dogs raccoon dogs and cats) and E. granulosus s.s. have been experimentally documented (Kapel et al., 2006) but are unknown for other Echinococcus spp.

• Mechanism of pathogenicity

  The pathogenicity depends on the parasite and host species, location of the metacestodes in the body and its size. Expansive (CE) or invasive (AE) growth of the metacestode can cause organ failure and systemic implications. There is a delimited inflammatory reaction in the surrounding tissue, with formation of a fibrous, encapsulating membrane. The parasite laminated wall of the cyst or macrovesicles may calcify, the cyst/lesion may then remain asymptomatic (Kern et al. 2017).

  GAP:

  Immunological reactions against the metacestode have been well studied but more research is needed to elucidate the immunomodulation properties of the parasite.

• Zoonotic potential

• Reported incidence in humans

  Detailed data for Europe: see Deplazes et al. (2017). All in all, in Europe yearly several thousands of new CE cases can be estimated, for AE around 200 new cases can be expected.CE: In Europe, data on human CE are fragmentary due to the lack of dedicated reporting and documentation systems. A European register is in the process of being established (Rossi et al., 2016). Sporadic CE cases, often imported, can be expected anywhere in Europe, but substantial numbers of cases are limited to Southern and Eastern Europe (Deplazes et al., 2017). Annual surgical incidence (per 100,000) can be as high as 9 (Northern Cyprus), 7 (Sardinia) and 4 (Sicily). In Europe, there is a general trend to reduced incidences, e.g. in Greece from 12.9 in 1984 to 0.25 in 2010 (Sotiraki et al. 2003). In the context of the recently completed EU project HERACLES, high CE prevalence (based on abdominal ultrasound screening surveys) was found among inhabitants of rural villages in southeastern Europe. Age/sex adjusted prevalence was 0.41% (95% CI 0.26-0.65) in Bulgaria, 0.41% (0.26-0.65) in Romania and 0.59% (0.19-1.85) in Turkey (Tamarozzi et al., 2018).

  AE: Despite missing efficient reporting systems, there is convincing evidence for an emergence of AE in the last decade in some European regions (Gottstein et al. 2015) In Switzerland, the incidence of infection increased from 0.1 to 0.26 / 100,000 / annum between 1993 and 2005 (Schweiger et al. 2007). In Austria, the total new cases per year varied between 2.4 and 2.8 in the period 2001 to 2010 but in 2011, 13 new cases were registered (Schneider et al. 2013). A steady increase of cases was reported from Poland between 1990 and 2011 (Deplazes et al., 2017). In Lithuania, the AE incidence per 100,000 increased from 0.03 in 2004 to 0.74 in 2012 (Marcinkute et al. 2015).

  GAP :

  Reporting of CE and AE cases is missing in many European countries and underreporting may be significant if reported.Official EU, OIE, national records- are unreliable indicators of human CE, AE and livestock rates for CE.

• Risk of occurence in humans, populations at risk, specific risk factors

  For CE, 8 case-control studies and 13 cross-sectional studies were included in a meta-analysis (Possenti et al., 2016). Case-control studies originated from South America, North Africa/Middle East, Turkey and Spain. Cross-sectional studies from China/Central Asia, the Middle East / North Africa, sub-Saharan Africa, South America, Turkey and Greece were included in this meta-analysis. The authors conclude that ‘Results from case-control studies do not provide significant evidence that CE is a strictly food- or waterborne disease’ and, for the cross-sectional studies, that the risk of CE transmission through the ingestion of food and water contaminated with E. granulosus s.l. eggs was not evidence based and is potentially anecdotal’.

  For AE, a meta-analysis for potential risk factors has been conducted, including 6 case-control and 11 cross-sectional studies (Conraths et al., 2017). The authors conclude that “the chance of AE transmission through the ingestion of food and water contaminated with E. multilocularis eggs does indeed exist, but it is important to note that food- and waterborne potential risk factors do not significantly increase the risk of infection”. The case-control studies originated from central Europe (4), Alaska (1) and China (1), the cross-sectional studies came predominantly from China (10), only one from central Europe. As the epidemiology of AE in China and Alaska differs from Europe, the results from the European studies included in the meta-analysis are listed separately:a) Case-control studies. Four European studies are included in the meta-analysis, of which three deal with food-related potential risk factors:For example: Kern et al., 2004. A study from Germany based on 40 AE patients. High OR’s were related to dog ownership and farming activities (OR’s from 4.7 to 18.0). Any food-related factors (eating raw produce) gave OR’s <2.5 except ‘chewing grass’ (OR 4.4, 95% CI 1.7-11.2).- Piarroux et al., 2013. A study from France based on 180 AE patients, 164 of which lived in known AE endemic regions. The dominant risk factor for the latter was agricultural occupation (OR 7.33, 95% CI 3.13-20.00) Minor or no risks were associated with food (eating raw wild salads OR 1.80 95% CI 0.98-3.31); eating raw wild berries OR 0.95, 95% CI 0.33-2.50), although ‘having a kitchen garden’ had an OR of 5.50 (95% CI 2.52-12.66).

  GAP:

  There are no data specifying the contribution of food consumer’s practices. Certain consumer behaviour life style or working conditions may increase the risk of exposure to Echinococcus spp. For example, regular close contact with dogs or activities in agriculture or in private gardens have been claimed to be associated to increased infection risks. Risks based on contamination of food in production or in the kitchen with egg contaminated substrates or surfaces have so far not been identified. Furthermore, inappropriate washing of vegetables or fruits may contribute to a higher exposure with Echinococcus eggs, but again, no data are available documenting that for example a higher exposure to E. multilocularis eggs will result in higher incidences of disease.

• Symptoms described in humans

  Variable clinical signs in humans depending on organ localization: CE and AE may remain silent for years before clinical symptoms become apparent. Rupture of the cysts can produce an acute disease with fever, urticaria, eosinophilia and anaphylactic shock as well as cyst dissemination. In addition to the liver and lungs, other organs (brain, bone, heart) can also be involved, with resulting symptoms. AE affects mainly the liver. The slow growing, destructive and invasive tumour-like lesion may cause abdominal pain, biliary obstruction, and occasionally metastatic lesions into the lungs and brain, but can develop over years without symptoms.

  GAP:

  Mass screening methods non-invasive early detection methods are urgently needed. Ultrasound is the most sensitive method but can only be applied in certain screening studies on population level. Improved serological tests would be of major interest.

• Estimated level of under-reporting in humans

  Worldwide including EU, reporting of cases of AE and CE is not well established and therefore underreporting has to be considered.

  In Europe, data on human CE are fragmentary due to the lack of dedicated reporting and documentation systems. A European register is in the process of being established (Rossi et al., 2016). Human AE is not a reportable disease in many EU member states. This needs to be changed: EFSA, Echinococcus multilocularis infection in animals. EFSA Journal 2015;13(12):4373.

  GAP :

  Major problems with reporting are: missing differentiation between CE and AE.

  Only in Germany an evaluation of the reporting system has been performed. According to this, the notification system failed to detect 67% of AE cases over a 3-year period (Jorgensen et al. 2008). Therefore, in many areas (e.g. France, Germany) sound baseline data are missing for a statistically based documentation of increased AE incidences.

• Likelihood of spread in humans

  Person to person spread does not occur.

• Impact on animal welfare and biodiversity

• Both disease and prevention/control measures related

  Usually none.

• Endangered wild species affected or not (estimation for Europe / worldwide)

  Europe: fatal alveolar echinococcosis in captive zoo mammals. E. multilocularis in translocated mammals for re-population of extinct habitats e.g. beavers in the UK.

  Worldwide: Possibly e.g. fatal cystic echinococcosis in wild wallaby species in Australia.

• Slaughter necessity according to EU rules or other regions

  Not mandatory even controlled periodic slaughtering of old animals (sheep over 6 years) in endemic countries could decrease transmission.

• Geographical distribution and spread

• Current occurence/distribution

  The geographic distribution of E. granulosus s.l. and E. multilocularis has been published recently (Deplazes et al. 2017).

  CE: E. granulosus s.l. occurs practically worldwide, and more frequently in rural, grazing areas. E. granulosus s.s. (G1-3) is prevalent in South and South East Europe, E. intermedius (G7) in the Baltic states, Poland and and is also focally distributed in South Europe (Portugal, Corsica, Italy, Greece).

  AE: E. multilocularis occurs in the northern hemisphere, including central Europe, Eurasia, and North America.

  High prevalence’s in foxes are recorded in Central and Eastern Europe including the Baltic area. The southern border of the endemic area is not exactly determined but until 2018 documented to be Central France, the Alps (Eastern France, Southern Switzerland, Northern Italy, Croatia, Serbia, Romania). In Northern Europe, Southern Sweden and Denmark and the Baltic states have been documented as endemic areas (so far no positive reports available from Norway and Finland. The most western endemic area is the Bretagne. GB and Ireland are free of E. multilocularis.

• Epizootic/endemic- if epidemic frequency of outbreaks

  In CE endemic areas the prevalence in sheep can be >60% and human prevalences >5%; prevalence in dogs may be >10%. Pastoral areas with livestock, dogs and people help maintain the transmission of E. granulosus s.s. through home slaughter and feeding of offal to dogs. Endemic areas of E. multilocularis may have high prevalence in foxes (>50%) and usually < 5% in small mammals; dogs prevalence <1% on population level, but can reach >10% in animals with free access to infected rodents (farm dogs).

  GAP:

  Spatial dynamic (stability or rate of spread) of E. multilocularis in Central and Northern Europe not well documented.

• Seasonality

  Not well defined in E. granulosus s.l. however, in the northern hemisphere home slaughter occurs mostly during the cold seasons. Home slaughter rates can also increase during religious festivals. In Sardinia all year round but especially after the lactation season (June) and after birth for very old sheep (February).For E. multilocularis fox predation on small mammals occurs primarily in autumn/winter thus facilitating most transmission.

  GAP:

  Optimal targeting of interventions e.g. dog treatment during or after seasonal periods when livestock slaughter is at its highest; and when cultural /religious perennial slaughter practices occur.

• Speed of spatial spread during an outbreak

  Outbreaks have so far not been documented for Echinococcus spp. Long distance movement of wild carnivores could contribute to the spread of the parasites. However, endemisation of the species is probably very slow based on transmission dynamics involving intermediate hosts.

• Transboundary potential of the disease

  Potentially spread via dogs or wildlife excreting eggs in faeces; to a lesser degree by movement/importation of live animals for slaughter.

  GAP:

  Passport for PETS schemes –how effective are they; should they be increased in Europe?Pets are not a problem for CE, at least in Sardinia, the real problem are shepard and stray dogs but they don’t travel. Adoption dogs are also not a problem as they are normally treated before. New trends in pet food use, such as BARF, could represent new risks for CE transmission.

  Consolidation phase of control of E. granulosus may require restriction of dog and livestock movement-how long?

• Route of Transmission

• Usual mode of transmission (introduction, means of spread)

  The intermediate hosts (IH) (including dead-end hosts like humans) become infected by ingesting eggs with resulting release of oncospheres in the intestine and the development of cysts/lesions in various organs. The definitive host (DH) becomes infected by ingesting the cyst-containing meat or viscera of the infected intermediate host. In the case of E. granulosus sheep and other intermediate hosts such as cattle acquire infections by grazing on pastures contaminated with dog faeces containing eggs. Dogs are, in turn, infected by ingesting meat or viscera containing such cysts, for example by eating an infected rodent (for E. multilocularis).

  Both intermediate or definite hosts and eggs of Echinococcus spp. with contaminated matrix can be responsible for first introduction of Echinococcus spp. However, for the parasites to become endemic, susceptible intermediate and definitive hosts and transmission between these hosts have to be guaranteed. For example in UK both susceptible foxes and predated susceptible voles are present (therefore UK is at risk of endemisation of AE after an introduction of infected IH or DH including pet dogs.

  GAP :

  In many E. multilocularis areas the key intermediate hosts maintaining the cycle are not identified.

• Occasional mode of transmission

  None

• Conditions that favour spread

  High populations of stray and shepard dogs.Home slaughtering activities and free access of dogs to viscera; free roaming dogs may scavenge carcasses or predate on small mammals.

  High fox populations especially in urban areas with access to vole habitats.

  GAP :

  Dogs in risk being infected with Echinococcus spp. should be dewormed regularly (see www.esccap.org for guidance.

• Detection and Immune response to infection

• Mechanism of host response

  Humoral and cellular immunological reactions can be documented in E. granulosus s.s. infected dogs, however these reactions were not of diagnostic value (low sensitivity and specificity) and none of these immune responses has been associated with protection against the parasite.

  In livestock anti-oncospheral antibodies after egg exposure can kill invading oncospheres. This is extremely effective in preventing subsequent re-infections (concomitant immunity); post-encystment immunity appears to be cell-mediated but less effective and poorly quantified.

  GAP :

  So far, no immunological protective mechanisms have been documented in DH and the claimed protective effects of experimental vaccination of dogs has been discussed controversially.

• Immunological basis of diagnosis

  Serological diagnosis of specific antibodies in definitive hosts is not of practical value. Coproantigens can be detected in definitive hosts infected with E. granulosus or E. multilocularis.Antibodies directed against native metacestode antigens (cyst fluid and protoscolex antigens) can be detected in the serum of infected IH. Because of low sensitivity and specificity such test has so far not been used in control programmes.

  GAP:

  No commercialised tests are available for serology in animals or coproantigen detection in the DH.

• Main means of prevention, detection and control

• Sanitary measures

  Current concepts for control are different for AE and CE, as AE is caused by a parasite with a predominantly wild animal lifecycle, whereas CE is caused in Europe by parasites of domesticated animals.CE: Sanitary measures to break the cycle between dog and sheep have been implemented in all control programs in the past.AE: Baiting of foxes and treatment of dogs are the only methods to minimise the environmental contamination with E. multilocularis eggs. General recommendations have been proposed but are difficult to justify and quantify.

  GAP :

  General recommendations concerning sanitary measures especially in CE caused by E. granulosus s.s. did not prove to be effective in controlling CE transmission in the European endemic area.No methods are available to estimate the major ways of transmission for both CE and AE and there is no scientific evidence that general recommendations have a preventing effect.

• Mechanical and biological control

  CE: Domestically transmitted species of the E. granulosus s.l. complex are, in theory, sensitive to control measures. As transmission depends on dogs having access to slaughter offal, complete supervision of slaughtering practices by qualified personnel, such that dogs do not have access to slaughter offal, should be sufficient to interrupt the lifecycle. This has been achieved through improved general slaughtering hygiene (and without any specific control measures implemented) in large parts of central and western Europe, where CE is now reduced to sporadic occurrence (Deplazes et al., 2017).

  Where a high-standard slaughtering is difficult to achieve, where home slaughtering is common or, where owned or stray dogs have access to dead animals on pastures, alternative measures should be applied (registration and regular deworming of dogs, education of stakeholders). By such approaches, CE has been eradicated under island conditions (Iceland, New Zealand and Tasmania). For such targeted long-term control programmes, detailed protocols are available, providing recommendations for consecutive project phases spanning >30 years (planning / attack / consolidation / maintenance phase) (Craig et al., 2017).

  AE: Control of E. multilocularis is currently not being done as a routine measure. However, numerous experimental studies have shown that effective control can be achieved temporarily by reducing infection in wild foxes. This can be achieved through application of baits containing anthelmintic agents (praziquantel). Protocols have been published with variable parameters concerning bait density (up to 50 per km2), baiting frequency (1/month to 2/year) and methods of distribution (by aircraft, by car, by hand) and inclusion or exclusion of human settlements. Control by praziquantel baiting has been tested in large rural areas (up to 5000 km2), as well as in small, circumscribed areas of urban environment. Tools for control have been developed, are ready for use and can be adapted to different environments (Hegglin and Deplazes, 2013; Craig et al., 2017). Regular anthelmintic treatment of dogs is proposed as an additional measure, in particular to decrease transmission probability to humans.

  GAP:

  National or large regional control programs are needed with strict sanitary measures supervised by official VPH institutions. Control of stray dog populations is a challenge in all programmes based on regulations of animal welfare and other regulations. Especially in extensive sheep farming including many small and fragmented units it is extremely difficult to reach more than 75% of the dogs for the regular deworming (frequency is depending on local factors but varies between 4 and 10 times/year).

• Diagnostic tools

  Diagnosis of echinococcosis in dogs or other susceptible carnivores relies on the demonstration of adult cestodes of the Echinococcus genus in the small intestine after necropsy or after arecoline purgation. Recently, coproantigen and copro-DNA assays have proven useful for safe, fast and accurate diagnosis. In intermediate hosts, diagnosis of E. granulosus depends on detection of the larval cyst form particularly the liver and lungs. Identification of small cysts/lesions may require histology or PCR for DNA analysis.

  GAP:

  Improved copro-PCR sensitivity needed DNA /PCR tests for E. granulosus genotypes useful for epidemiology.

  Use of DNA markers to identify Echinococcus isolates by region; indication of spread for E. multilocularis.

• Vaccines

  There are currently no effective drugs or vaccines to protect humans against the disease. Progress has been made in the development of an effective anti-oncosphere recombinant vaccine (EG95) against egg infection with E. granulosus in sheep and cattle. (4). Experimental vaccines for dogs have been investigated but not available and their results highly controversial and not satisfactory.

  GAP:

  What is potential for a dog vaccine for E. granulosus and a fox vaccine for E. multilocularis? Difficult to set up experiments; anti-fecundity vaccines rather than anti-establishment adult worm vaccines.

• Therapeutics

  Human disease is usually treated by surgical removal of the cyst with supplementary chemotherapy (Mebendazole or Albendazole). Chemotherapy alone may have < 35% cure rates with less efficacy for alveolar echinococcosis. All dogs, especially those in rural endemic areas should be treated 4-8 times per year with a wormer containing Praziquantel.

  GAP:

  Therapeutic options for CE1-CE5 different pathological presentations. Albendazole alone or with surgery.Efficacy of ABZ and praziquantel in combination for human CE?Efficacy of Oxfendazole in livestock CE and human trials?

• Biosecurity measures effective as a preventive measure

  Prevent dogs having access to infected viscera; proper disposal of livestock carcasses; requirement of P3/P4 containment in research facilities especially where fox/dog necropsy and/or faecal handling occurs.

• Border/trade/movement control sufficient for control

  Possible/advisable treatment of dogs with praziquantel before movement in some circumstances especially to avoid introducing Echinococcus species into free regions

  GAP:

  Passport for PETS schemes

• Prevention tools

  The advent of a new vaccine (EG95) for livestock may help reduce the time required to interrupt transmission between dogs and sheep and the risk of human exposure.

  GAP:

  Need to show whether EG95 vaccine for ovine CE in combination with dog praziquantel dosing can decrease years of control required for hydatid disease. How sustainable? Need proof of principle 5-year trial in highly endemic region(s).

• Surveillance

  CE: Detecting cysts of E. granulosus at meat inspection, thus targeting infected farms or communities. Diagnosis of dog transmitted taeniids (T. hydatigena and E. granulosus) by coproantigen ELISA, but also by conventional microscopical egg detection in faeces and genetic analysis of the taeniid eggs.

  AE: Necropsy surveys for E. multilocularis in foxes or coproantigen or genetic analysis of environmental faecal samples to determine the contamination with E. multilocularis eggs....

  GAP :

  Livestock slaughter inspection records (from local slaughterhouses or purchased animals) remains the gold standard, but cysts <1 cm require histological verification or DNA analysis.

  No commercialised coproantigen tests are available. Genetic tools are available only in specialised laboratories and are expensive.

  Livestock serology is of limited use—probably only for imported or exported live animals and possibly for herd testing for consolidation or maintenance of eradication phases

• Past experiences on success (and failures) of prevention, control, eradication in regions outside Europe

  Control programs for cystic echinococcosis can be successful but require a long period of intervention (>5-10 years) based primarily on dog-targeted control measures. (1). Temporary reduction in transmission of E. multilocularis can be achieved using praziquantel baits for foxes.

  GAP:

  Integrated use of EG95 vaccine with other measures (subsidised slaughter of old sheep bearing the main part of E. granulosus biomass in a population) for more rapid and effective control of CE.

• Costs of above measures

  CE: Expensive with the use of deworming drugs for high cover rates over extended periods.

  AE: High cost of control contrasts with comparatively low numbers of AE patients in Europe, requiring risk management decisions (Hegglin and Deplazes, 2013).

  GAP:

  Requires logistics, manpower and long time commitment over decades.

• Disease information from the WOAH

• Disease notifiable to the WOAH

  YES

• WOAH disease card available

  No.

  GAP :

  Not applicable

• WOAH Terrestrial Animal Health Code

  http://www.oie.int/index.php?id=169&L=0&htmfile=chapitre_echinococcus_granulosus.htmhttp://www.oie.int/index.php?id=169&L=0&htmfile=chapitre_echinococcus_multilocularis.htm

  GAP :

  Not applicable

• WOAH Terrestrial Manual

  http://www.oie.int/fileadmin/Home/eng/Health_standards/tahm/3.01.06_ECHINOCOCCOSIS.pdf

  GAP :

  Not applicable

• Socio-economic impact

• Zoonosis: impact on affected individuals and/or aggregated DALY figures

  Echinococcosis is a serious zoonosis, with rates of human E. granulosus cystic echinococcosis infection ranging from less than 1 per 100,000 to more than 200 per 100,000 in certain rural populations where there is close contact with domestic dogs. Incidence of human alveolar echinococcosis caused by E. multilocularis is usually < 0.5 per 100,000 but may be >100 per 100,000 in certain communities (e.g. Tibetan herdsmen). (1). Total global burden of cystic echinococcosis > 1 million DALYs; for alveolar echinococcosis >650,000 DALYs.

• Zoonosis: cost of treatment and control of the disease in humans

  Surgery and the long term use of chemotherapy with parasiticides are expensive.

  GAP:

  Differential treatment approach based on CE1-CE5 pathological status; Watch and Wait approach; tendency to use surgery before medical treatment with ABZ

• Direct impact (a) on production

  Small. But livestock production may decrease by approx 5% in endemic E.granulosus areas.

  GAP:

  Are there clinical signs in livestock CE infections?

• Direct impact (b) cost of private and public control measures

  High cost of praziquantel, and dog/fox dosing teams/efforts. Potential costs of vaccines are developed for use to prevent infection by larvae

  GAP:

  Integrated control of more than one zoonosis e.g. CE with leishmaniosis, brucellosis.One Health approach for low-resource populations.

• Indirect impact

  Main loss is in the condemnation of offal (liver/lungs) due to the presence of hydatid cysts; cost of dewormers by dog owners.

  GAP :

  Role of urban foxes in transmission and risk of AE in Europe.

• Trade implications

• Impact on international trade/exports from the EU

  None but the OIE recommends that Veterinary authorities of the importing country should require the presentation of a international veterinary certificate attesting that dogs, cats and other domestic or wild carnivores were treated against echinococcosis/hydatidosis prior to shipment, and that the treatment used is recognised as being effective. Importation of dogs/cats into some E.multilocularis-free countries of EU requires a pet passport with certified evidence of praziquantel dosing 48-70hrs prior to importation.

  GAP:

  Continued use of PET schemes in Europe for prevention E. multilocularis spread; harmonisation across Europe-threat? Prevention of E. multilocularis spread in Europe?EU scheme for preventive health measure (Regulation (EU) No 1152/2011).

• Impact on EU intra-community trade

  None, but the procedures described under section “Impact on international trade/exports” is required for import into certain countries of the EU.

• Impact on national trade

  None

• Main perceived obstacles for effective prevention and control

  The best control measure is to interrupt the life cycle of the parasite. Relatively easy )though long-term) with E.granulosus but more difficult with E. multilocularis due to the cycle in wildlife through foxes and rodents

  GAP:

  Improved integrated control for CE praziquantel bait for E. multilocularis in foxes and E. multilocularis vaccine baits for fox infection.

• Main perceived facilitators for effective prevention and control

  • Regular dosing of dogs with praziquantel for E.granulosus prevention• Reduction in transmission has been achieved by use of praziquantel baits for foxes and dosing of owned dogs where spill-over into the dog population occurs in the case of E.multilocularis. • Vaccination of sheep (or other livestock) to protect against the development of the larval stage of E. granulosus.

  GAP :

  Specified dog dosing frequency 2-12 times per year? Optimisation of dosing rates?

  Role of sustained dog dosing for Em infections in Central Asia

  Test use of EG95 vaccine and PZQ dosing 2x per year VS PZQ alone etc

• Links to climate

  Seasonal cycle linked to climate

  No

• Distribution of disease or vector linked to climate

  No

• Outbreaks linked to extreme weather

  CE: Possibly associated with periods of drought or cold, resulting in higher than normal livestock deaths with resultant increase in scavenging by dogs

  GAP:

  Droughts and severe winters may increase livestock deaths in the field and risk of scavenging by dogs/canids.

• Sensitivity of disease or vectors to the effects of global climate change (climate/environment/land use)

  Changes in land use can affect the populations of susceptible hosts of E. granulosus or E. multilocularis. Habitat changes based on climate change could contribute to the increase/decrease of susceptible vole species for E. multilocularis (e.g. expansion to the north in Scandinavia).

  GAP :

  Long-term ecological investigations needed.

Risk

• Major neglected zoonoses for the developing world.Impact on human health in the developing world where dogs are not wormed and the cycle of E. granulosus is allowed to continue. Gradual spread of E.multilocularis into Western Europe poses risks to the population.

Conclusion

• Human cystic echinococcosis (caused by E. granulosus s.l.) and alveolar echinococcosis (caused by E. multilocularis) are important public health threats in many parts of the world including most parts of Europe. The gradual increase of E. multilocularis infection pressure due to the increasing fox populations and the spread to Western, Northern and Eastern Europe is causing concern. Some areas in Europe have an incidence of CE in humans among the highest in the world. There is an urgent need to begin new, active steps to reduce this burden on human health. The necessary tools and knowledge are both available to control the disease. What is needed is European manufacture and registration of the EG95 vaccine for livestock and political will and funding to undertake control programs.For E. granulosus s.l. the best control measure is to interrupt the domestic life cycle of the parasite. This can be achieved with well-designed, integrated control programs based on deworming of dogs and vaccination of lambs over the long-term. Control of E. multilocularis is more complex because the cycle occurs within wildlife. The development of (i) improved diagnostic tests to use in monitoring of control programmes and for the individual fast diagnosis in dogs and (i) improved methods to control the infection in wildlife are needed.

  GAP :

  Priorities in national actions required, long-term actions and secure funding for control and monitoring (over several decades) have to be considered.

Sources of information

• Expert group composition

  Peter Deplazes, University of Zürich, Switzerland - [Leader]

  Antonio Varcasia, UNISS, Italy

  Franck Boué, ANSES, France

  Smaro Sotiraki, HAO Demeter, Greece

  Marshall Lightowlers, University of Melbourne, Australia

  Conraths, Franz Josef, Friedrich Loeffler Institute, Germany

• Reviewed by

  Project Management Board

• Date of submission by expert group

  30 March 2019

• References

  Craig, P.S., Hegglin, D., Lightowlers, M.W., Torgerson, P.R. and Wang, Q., 2017. Echinococcosis: control and prevention. In Advances in parasitology (Vol. 96, pp. 55-158). Academic Press.

  Deplazes, P., Rinaldi, L., Rojas, C.A., Torgerson, P.R., Harandi, M.F., Romig, T., Antolova, D., Schurer, J.M., Lahmar, S., Cringoli, G. and Magambo, J., 2017. Global distribution of alveolar and cystic echinococcosis. In Advances in parasitology (Vol. 95, pp. 315-493). Academic Press.

  EFSA BIOHAZ Panel (EFSA Panel on Biological Hazards), 2015. Scientific opinion on the evaluation of heat treatments, different from those currently established in the EU legislation, that could be applied to live bivalve molluscs from B and C production areas, that have not been submitted to purification or relaying, in order to eliminate pathogenic microorganisms. EFSA Journal 2015;13(12):4332, 76 pp. doi:10.2903/j.efsa.2015.4332

  Gottstein, B., Stojkovic, M., Vuitton, D.A., Millon, L., Marcinkute, A. and Deplazes, P., 2015. Threat of alveolar echinococcosis to public health–a challenge for Europe. Trends in Parasitology, 31(9), pp.407-412.

  Hegglin, D. and Deplazes, P., 2013. Control of Echinococcus multilocularis: strategies, feasibility and cost–benefit analyses. International journal for parasitology, 43(5), pp.327-337.

  Jorgensen, P., An der Heiden, M., Kern, P., Schöneberg, I., Krause, G. and Alpers, K., 2008. Underreporting of human alveolar echinococcosis, Germany. Emerging Infectious Diseases, 14(6), p.935.

  Kapel, C.M.O., Torgerson, P.R., Thompson, R.C.A., Deplazes, P., 2006, Reproductive potential of Echinococcus multilocularis in experimentally infected foxes, dogs, raccoon dogs and cats. Int J Parasitol 36, 79-86.

  Kern, P., da Silva, A.M., Akhan, O., Müllhaupt, B., Vizcaychipi, K.A., Budke, C. and Vuitton, D.A., 2017. The echinococcoses: diagnosis, clinical management and burden of disease. In Advances in parasitology (Vol. 96, pp. 259-369). Academic Press.

  Marcinkutė, A., Šarkūnas, M., Moks, E., Saarma, U., Jokelainen, P., Bagrade, G., Laivacuma, S., Strupas, K., Sokolovas, V. and Deplazes, P., 2015. Echinococcus infections in the Baltic region. Veterinary parasitology, 213(3-4), pp.121-131.

  Possenti, A., Manzano-Román, R., Sánchez-Ovejero, C., Boufana, B., La Torre, G., Siles-Lucas, M. and Casulli, A., 2016. Potential risk factors associated with human cystic echinococcosis: systematic review and meta-analysis. PLoS neglected tropical diseases, 10(11), p.e0005114.

  Romig, T., Deplazes, P., Jenkins, D., Giraudoux, P., Massolo, A., Craig, P.S., Wassermann, M., Takahashi, K. and De La Rue, M., 2017. Ecology and life cycle patterns of Echinococcus species. In Advances in parasitology (Vol. 95, pp. 213-314). Academic Press.

  Schneider, R., Aspöck, H. and Auer, H., 2013. Unexpected increase of alveolar echincoccosis, Austria, 2011. Emerging Infectious Diseases, 19(3), p.475.

  Schweiger, A., Ammann, R.W., Candinas, D., Clavien, P.A., Eckert, J., Gottstein, B., Halkic, N., Muellhaupt, B., Prinz, B.M., Reichen, J. and Tarr, P.E., 2007. Human alveolar echinococcosis after fox population increase, Switzerland. Emerging infectious diseases, 13(6), p.878.

  Sotiraki, S., Himonas, C. and Korkoliakou, P., 2003. Hydatidosis–echinococcosis in Greece. Acta tropica, 85(2), pp.197-201.

  Tamarozzi, F., Akhan, O., Cretu, C.M., Vutova, K., Akinci, D., Chipeva, R., Ciftci, T., Constantin, C.M., Fabiani, M., Golemanov, B. and Janta, D., 2018. Prevalence of abdominal cystic echinococcosis in rural Bulgaria, Romania, and Turkey: a cross-sectional, ultrasound-based, population study from the HERACLES project. The Lancet Infectious Diseases, 18(7), pp.769-778.