Untargeted metabolomics to discriminate liver and lung hydatid cysts: Importance of metabolites involved in the immune response.

The Echinococcus granulosus sensu lato species complex is responsible for the neglected zoonotic disease known as cystic echinococcosis (CE). Humans and livestock are infected via fecal-oral transmission. CE remains prevalent in Western China, Central Asia, South America, Eastern Africa, and the Mediterranean. Approximately one million individuals worldwide are affected, influencing veterinary and public health, as well as social and economic matters. The infection causes slow-growing cysts, predominantly in the liver and lungs, but can also develop in other organs. The exact progression of these cysts is uncertain. This study aimed to understand the survival mechanisms of liver and lung CE cysts from cattle by determining their metabolite profiles through metabolomics and multivariate statistical analyses. Non-targeted metabolomic approaches were conducted using quadrupole-time-of-flight liquid chromatography/mass spectrometry (LC-QTOF-MS) to distinguish between liver and lung CE cysts. Data processing to extract the peaks on complex chromatograms was performed using XCMS. PCA and OPLS-DA plots obtained through multiple statistical analyses showed interactions of metabolites within and between groups. Metabolites such as glutathione, prostaglandin, folic acid, and cortisol that cause different immunological reactions have been identified both in liver and lung hydatid cysts, but in different ratios. Considering the differences in the metabolomic profiles of the liver and lung cysts determined in the present study will contribute research to enlighten the nature of the cyst and develop specific therapeutic strategies.

Safety of Three-Dimensional Conformal Radiotherapy on the Sheep with Pulmonary Cystic Echinococcosis: A Preliminary Study.

PURPOSE: Radiotherapy showed the potential to effectively kill the cysts of pulmonary cystic echinococcosis (CE). However, little is known about its safety. This study was designed to investigate the safety of three-dimensional conformal radiotherapy (3D-CRT) on the normal lung tissue adjacent to the cyst and blood of sheep naturally infected with pulmonary CE. METHODS: Twenty pulmonary CE sheep were randomly divided into control group (n = 5) and radiation groups with a dose of 30 Gray (Gy) (n = 5), 45 Gy (n = 5), and 60 Gy (n = 5), respectively. Animals in control group received no radiation. Heat shock protein 70 (Hsp70), tumor growth factor-ß (TGF-ß), matrix metalloproteinase-2 (MMP-2) and MMP-9 in the lung tissues adjacent to the cysts, which were considered to be closely related to the pathogenesis of CE, were evaluated after 3D-CRT. A routine blood test was conducted. RESULTS: The results showed that there were multiple cysts of various sizes with protoscoleces in the lung tissues of sheep, and necrotic cysts were found after 3D-CRT. 3D-CRT significantly increased the mRNA level of Hsp70, enhanced the protein level of TGF-ß and slightly increased the expression of MMP-2 and MMP-9 in lung tissues adjacent to the cysts. 3D-CRT did not significantly alter the amount of WBC, HB and PLT in sheep blood. CONCLUSIONS: The results suggested that 3D-CRT may suppress the inflammation and induce less damage of the normal lung tissues and blood. We preliminarily showed that 3D-CRT under a safe dose may be used to treat pulmonary CE.

Response patterns in adventitial layer of Echinococcus granulosus sensu stricto cysts from naturally infected cattle and sheep.

Cystic echinococcosis is a zoonotic disease caused by the metacestode of Echinococcus granulosus sensu lato. The disease is characterized by the development of cystic structures inside viscera of the intermediate host, mainly liver and lungs. These cysts are formed by three layers: germinal, laminated, and adventitial layer, the latter being the local host immune response. Metacestodes that develop protoscoleces, the infective stage to the definitive host, are termed fertile, whereas cysts that do not produce protoscoleces are termed non-fertile. Sheep usually harbor fertile cysts while cattle usually harbor non-fertile cysts. Adventitial layers with fibrotic resolution are associated to fertile cysts, whereas a granulomatous reaction is associated with non-fertile cysts. The aim of this study was to analyze cellular distribution in the adventitial layer of fertile and non-fertile E. granulosus sensu stricto cysts found in liver and lungs of cattle and sheep. A total of 418 cysts were analyzed, 203 from cattle (8 fertile and 195 non-fertile) and 215 from sheep (64 fertile and 151 non-fertile). Fertile cysts from cattle showed mixed patterns of response, with fibrotic resolution and presence of granulomatous response in direct contact with the laminated layer, while sheep fertile cysts always displayed fibrotic resolution next to the laminated layer. Cattle non-fertile cysts display a granulomatous reaction in direct contact with the laminated layer, whereas sheep non-fertile cysts display a granulomatous reaction, but in direct contact with the fibrotic resolution. This shows that cattle and sheep cystic echinococcosis cysts have distinct local immune response patterns, which are associated to metacestode fertility.

Fasciola hepatica coinfection modifies the morphological and immunological features of Echinococcus granulosus cysts in cattle.

Polyparasitism occurs when animals harbour multiple parasites concomitantly. It is a common occurrence but is generally understudied in wild and domestic animals. Fasciola hepatica and Echinococcus granulosus, which are helminths of ungulates, frequently coinfect cattle. The effects of this particular type of polyparasitism are not well documented. The metacestode of Echinococcus granulosus is surrounded by the adventitial layer, which constitutes the host immune response to the parasite. This layer in cattle is produced by a granulomatous reaction and is involved in echinococcal cyst (EC) fertility. Due to the systemic immune-modulating abilities of Fasciola hepatica, coinfection possibly generates a favourable environment for EC growth. A total of 203 Echinococcus granulosus sensu stricto cysts were found in 82 cattle, of which 42 ECs were found in 31 animals coinfected with Fasciola hepatica. The overall infection intensity was 3 cysts per animal. Coinfection with Fasciola hepatica decreased the mean infection intensity to 1.4 cysts per animal. Regarding EC size, coinfection resulted in smaller ECs (15.91 vs 22.09 mm), especially for infertile lung cysts. The adventitial layer of ECs in coinfected animals lacked lymphoid follicles and palisading macrophages, which are generally hallmarks of the granulomatous immune response. The ECs in coinfected animals had organized laminated layers, whereas those in animals without coinfection did not. Although coinfection was not statistically associated with EC fertility, we did not find fertile cysts in the livers of coinfected animals. We concluded that coinfection with Fasciola hepatica and Echinococcus granulosus has a detrimental effect on ECs, particularly infertile cysts.

Molecular survey on cattle and sheep hydatidosis and first detection of Echinococcus canadensis (G6/G7) in sheep in Turkey.

Larval stage of genus Echinococcus is the causing agent for the zoonotic infection which is life threatening known as Echinococcosis. The purpose of this study was the identification, molecular analysis and characterization of Echinococcus spp. in sheep and cattle. The sampling was done from slaughterhouse of Elazig, Turkey. A total of 85 isolates (sheep, n = 19 and cattle, n = 66) have been collected after slaughtering. Following the gDNA isolation and PCR products of mt-CO1 gene (446 bp) of all the samples were sequenced. Out of 85 isolates, 84 were recognized as Echinococcus granulosus sensu stricto and one sheep isolate was found as Echinococcus canadensis (G6/G7 ) which is identified for the first time in Turkey. However, single nucleotide polymorphism has been observed not only in samples of different animals but also in samples collected from the same cattle. Six liver and three lung hydatid cysts have been detected in cattle. Although no nucleotide differences have been observed in the liver samples, there was single nucleotide polymorphism (C→T) in 40th nucleotide of two lung cysts. As a result of haplotype analysis, 16 haplotypes of E. granulosus s.s. were detected in 66 cattle isolates whereas 7 haplotypes of E. granulosus s.s. were identified in 19 sheep samples.

Pulmonary Echinococcus multilocularis metastasis in a dog.

A young adult Labrador retriever dog was presented for surgical debulking of hepatic alveolar echinococcosis. Computed tomography detected hepatomegaly with multiple large cavitary masses with extension of tissue from a lesion wall into the caudal vena cava and numerous nodules in all lung lobes. Following euthanasia, histology confirmed parasitic vesicles with granulomatous reaction in all lesions, and polymerase chain reaction (PCR) established the causative agent to be Echinococcus multilocularis. This report is the first to present imaging features of pulmonary E. multilocularis granulomata in a dog.

Métastases pulmonaires d'Echinococcus multilocularischez un chien. À l'examen par tomodensitométrie d'un Labrador retriever jeune adulte référé pour résection de lésions hépatiques d'échinococcose alvéolaire, une hépatomégalie avec présence de larges masses cavitaires fut mise en évidence, de même que l'extension de la paroi d'une lésion à l'intérieur de la veine cave caudale, et de nombreux nodules pulmonaires. Après euthanasie, des vésicules parasitiques associés à une réaction granulomateuse furent confirmés histologiquement dans toutes les lésions évaluées, et E. multilocularis fût démontré par PCR être l'agent causal. Ce rapport de cas est le premier à présenter les caractéristiques de lésions pulmonaires d'E. multilocularis chez le chien.(Traduit par les auteurs).

Oxidant/antioxidant status in lambs and sheep with liver and lung cystic echinococcosis diagnosed by ultrasonography and necropsy.

The aim of this study was to evaluate total antioxidant capacity (TAC), total oxidant status (TOS), and oxidative stress index (OSI) in sheep and lambs with cyctic eccinocoocosis (CE) diagnosed by ultrasonography and necropsy findings. A total of 9 sheep and 17 lambs with CE were used in this study and the findings were compared to those of 6 healthy control sheep. Ultrasonography were used for the diagnosis of CE in sheep and lambs, and necropsy was performed to check the presence of cysts in liver and lungs. Serum TOS and TAC were measured by a novel colorimetric method. The TOS-to-TAC ratios were also calculated as OSI values. Serum biochemical profiles were determined by conventional measurement methods as well. The mean values for TOS, TAC and OSI were significantly (p<0.001) lower in sheep and lambs with CE when compared with those of the control sheep, and they were also significantly lower in lambs with CE in comparison to the mean values obtained in sheep with CE. The levels of serum albumin, total cholesterol, creatinine, and triglycerides in lambs with CE were found out to decrease significantly (p<0.001) when compared with those of both sheep with EC and the control group. There were no significant differences between the groups in terms of other serum parameters. In addition, when clinically and some biochemical values were evaluated, CE was found to be more severe in lambs than in sheep. It was concluded that although common diagnostic cyst detection is performed by postmortem examination, ultrasonography could successfully be used in conjunction with serum biochemical profile detection and serum TOS, TAC and OSI measurements for diagnosis of cysts in liver and lungs of severely infected living sheep and lambs. Serum albumin, total cholesterol, creatinine, total protein and triglycerides might be used as indicators in sheep and particularly in lambs for the diagnosis of CE.

Boiling sheep liver or lung for 30 minutes is necessary and sufficient to kill Echinococcus granulosus protoscoleces in hydatid cysts.

Proper disposal of carcasses and offal after home slaughter is difficult in poor and remote communities and therefore dogs readily have access to hydatid cysts containing offal from livestock, thus completing the parasite cycle of Echinococcus granulosus and putting communities at risk of cystic echinococcosis. Boiling livers and lungs which contain hydatid cysts could be a simple, efficient and energy- and time-saving way to kill the infectious protoscoleces. The aim of this study was to provide precise practical recommendations to livestock owners. Our results show that boiling the whole sheep liver and/or lung, with single or multiple hydatid cysts, for 30 min is necessary and sufficient to kill E. granulosus protoscoleces in hydatid cysts. Advertising on this simple rule in at-risk communities would be an efficient and cheap complement to other veterinary public health operations to control cystic echinococcosis.

Prevalence of hydatidosis in slaughtered animals in Iran.

OBJECTIVE: Hydatid cyst or cystic echincoccosis (CE) is an important medical and veterinary problem in the world, especially in Iran. Domestic intermediate hosts are a major reservoir for the disease in humans. The aim of this study was to determine the prevalence of hydatid cysts in slaughtered animals in Isfahan, central part of Iran. METHODS: In this cross-sectional study performed from 10 May 2009 to 10 May 2010, a total of 196,325 animals (89,651 sheep, 93,050 goats, 9,112 cattle and 4,512 calves) were inspected macroscopically for hydatid cysts. RESULTS: Prevalence rate of CE in sheep, goats, cattle and calf was 16.4%, 3.1%, 6.5% and 8.2%, respectively. In all cases, the prevalence in female cattle and sheep was more than in males (P < 0.001). There are significant seasonal pattern for hydatidosis only in sheep (P < 0.001) and the highest prevalence of cysts was seen in autumn and winter seasons. The fertility of cysts in the liver of sheep (77%) was higher than in lungs (47.9%), but was higher in lungs in cattle (44%). Most condemnation cases were seen in lung of sheep (27.1%). CONCLUSION: It appears that sheep are the most important intermediate hosts for E. granolusus in this area.

Prevalence of hydatidosis among slaughtered animals in Jeddah, Kingdom of Saudi Arabia.

This study was conducted over one year in the major North Jeddah abattoir to assess the current status of hydatidosis among slaughtered animals. The cysts count size, site of infection and fertility were conducted on randomly infected animals. A total of (91348) slaughtered animals; camels (541), cattle (615), goats (48370) and sheep (41822) were macroscopically and microscopically examined. The infection prevalence was 6.86%, 3.63%, 69.6% & 19.85% in camels, cattle, sheep and goats respectively. The liver was the predominant site of infection in all animals. The intensity of cysts differed significantly among all host species. Most of cysts were small in size and was found more in the liver than in the lung. There was a significant difference among host species in fertile cysts (P < 0.00). The higher percentage of fertile cysts was in goat (9.87%) and sheep (4.85%). The viability rate of protoscolces was significantly different in all examined animals with a higher rate among sheep and goats. These findings reflect the existence of the life cycle maintenance and the transmission of the cestode Echinococcus granulosus from definitive host (dogs) to intermediate hosts (camels, cattle, sheep and goats) in Jeddah.

Drug-induced apoptosis of Echinococcus granulosus protoscoleces.

Parasitic infection by Echinococcus granulosus in humans induces hydatidosis (echinococcosis), which is a zoonotic disease that seriously endangers public health. This study was to determine the status of cell apoptosis in the protoscoleces of E. granulosus, which were isolated from hydatid cysts in livers or lungs of sheep. Those protoscoleces were incubated with drugs (at the concentration of 1 mmol L(-1) H(2)O(2) and 5 mmol L(-1)dexamethasone) for 8 h, the apoptosis were examined by transmission electron microscopy and TUNEL assay, the expression of caspase-1 and caspase-3 were detected by immunohistochemistry and caspase-3 activity was detected by colorimetric assay. Our results have clearly demonstrated the presence of cell apoptosis in protoscoleces in the absence or presence of drug (H(2)O(2), dexamethasone) treatment, but drug-induced apoptosis rate, caspase-1 and caspase-3 expression levels were higher than no-drug induce and caspase-3 activity were significantly increasing. We found H(2)O(2) and dexamethasone can induce the cell apoptosis of protoscoleces. Our results implied the existence of a CED-3 like apoptosis gene in protoscolces and provide a rationale for further exploring the induction of apoptosis as non-surgical treatment method in treating this parasitic disease.

An abattoir-based study of hydatidosis in the dromedary (Camelus dromedarius) in Mashhad, Iran.

A 6-year retrospective study based on abattoir records was carried out to determine the prevalence of hydatidosis in dromedaries in Khorasan province in north-eastern Iran. Between 20 March 2004 and 19 March 2010, 25,255 dromedaries were slaughtered in the study area and the livers of 2791 (11.1%) and the lungs of 3289 dromedaries (13.2%) were discarded due to hydatidosis. The annual prevalence of liver condemnations due to hydatidosis decreased from 24.1% in 2004-2005 to 13.3% in 2009, and finally to 6.8% in 2010. The corresponding features for lung condemnation due to hydatidosis were relatively higher than liver, declining from 28.7% in 2004-2005 to 14.9% in 2009, and finally to 7.1% in 2010. Liver and lung condemnations due to hydatidosis were significantly higher in the spring. This could be attributed to various factors such as sources of slaughtered animals, changes in management practice and ecological factors. The present survey provides baseline data for the future monitoring of this potentially important parasitic disease in the region.

Comparative pathology of pulmonary hydatid cysts in macropods and sheep.

The development and appearance of hydatid cysts of Echinococcus granulosus in experimentally infected tammar wallabies (Macropus eugenii) and sheep during the period 9-17 months post-infection (mpi) were studied. Cysts of unknown age were also examined from mature, naturally infected sheep. The cysts grew more rapidly and became fertile within a shorter period in wallabies compared with sheep. Cysts from the wallabies were larger in absolute size and were larger relative to the size of the lungs. Microscopical examination revealed that wallaby hydatid cysts developed in small bronchioles. Hydatid cysts in the wallabies had a thicker germinal membrane, with more nuclei and a thicker laminated layer (LL), than hydatid cysts of similar age found in sheep. In contrast, the adventitial layer was thicker in the ovine cysts, comprising a hyalinized layer of degenerate collagen and necrotic cellular debris surrounded by a layer of granulation tissue that was largely absent from lesions in the wallabies. Multilocular cysts were present in sheep, but not in wallabies. The greater thickness of the germinal membrane in wallaby cysts suggests greater parasite activity, which may explain the more rapid growth rate in this host, whereas the thicker adventitial layer in sheep cysts may be restrictive to growth while simultaneously protecting the hydatid from the host immune response. These differences in the parasite-host relationship between macropods and sheep may reflect the relatively recent introduction of the parasite into Australia.

An abattoir-based study on the prevalence and economic losses due to cystic echinococcosis in slaughtered herbivores in Ahwaz, south-western Iran.

A 10-year (1998-2008) retrospective study was carried out to investigate the prevalence and long-term trend of hydatid disease in slaughtered herbivores in the large complex abattoir of Ahwaz (the capital of Khuzestan province, south-western Iran). A total of 3,583,417 animals including 2,815,982 sheep, 427,790 goats and 339,645 cattle were inspected macroscopically for hydatid cysts in the 10-year period, and overall 155,555 (4.24%) livers and 228,172 (6.37%) lungs were condemned. Cystic echinococcosis (CE) was responsible for 36.08% and 48.04% of total liver and lung condemnations, respectively. The prevalence of pulmonary hydatid disease in sheep, goats and cattle was 2.22, 5.43 and 6.99%, respectively; on the other hand, the prevalence of hepatic hydatid disease for those animals was 1.26, 2.57 and 2.80%, respectively. Data showed an overall downward long-term trend for CE in all livestock slaughtered during the study period (P < 0.01). Lung condemnation due to CE was significantly more common than liver condemnation for each animal separately (P < 0.001). The prevalence of liver and lung hydatidosis in sheep was significantly lower than that in other livestock (P < 0.001). The prevalence of hydatid disease recovered from the sheep, cattle and goats varied in different seasons, but there was no statistical difference between various seasons. The odds ratio of lung and liver condemnations due to hydatidosis showed a slightly different pattern in some years; however, the overall declining trend was still observed. The total annual economic loss incurred due to hydatidosis in all ruminants slaughtered at Ahwaz municipal abattoir was estimated to be US$459,659.6, based on the market prices in the year 2008. This number corresponds to a loss of US$300,620.4 for cattle, US$123,490.0 for sheep and US$35,549.2 for goats. The current results provide baseline data for the future monitoring of this potentially important disease in the region, and also suggest that a thorough investigation leading to a disease control strategy is required to reduce the economic and public health consequences of CE.

Echinococcus granulosus: biological comparison of cattle isolates from endemic regions of Argentina and Spain / Echinococcus granulosus: comparación biológica de aislados de bovinos de regiones endémicas de Argentina y España

In the present study we have compared cattle isolates of Echinococcus granulosus from Argentina and Spain. The aim was to compare and determine if there exist phenotypic and genetic differences within E. granulosus cattle isolates between an endemic area of Spain (where the disease is mainly restricted to a sheep-dog cycle) and an endemic area of Argentina (where cattle are the most abundant intermediate hosts). The Spanish samples were previously identified as G1 genotype. The Argentinean samples were also identified as G1, but some variants were found for the cytochrome c oxidase-1 (CO1) and NADH dehydrogenase-1 (ND1) mitochondrial genes. When comparing the cyst features and the morphology of the larval rostellar hooks in both regions, some differences were found. The morphometric analyses of the larval rostellar hooks showed the existence of two distinct clearly separated groups (one corresponding to the Argentinean samples and the other to the Spanish ones). In conclusion, there are some genetic and phenotypic differences within E. granulosus cattle isolates from Argentina and Spain. Probably these differences, more important from an epidemiological point of view, are related to different steps in the disease control in both countries. Further studies involving other epidemiological, morphometric and molecular data, including other types of livestock, would contribute to clarify and expand the present work.

El objetivo del presente trabajo fue determinar si existen diferencias fenotípicas y genéticas entre los aislados de Echinococcus granulosus de origen bovino provenientes de dos regiones geográficas donde la hidatidosis es endémica, una de España (donde predomina el ciclo perro-oveja) y una de Argentina (donde el bovino es el hospedador intermediario más importante). Las muestras españolas fueron previamente identificadas como pertenecientes al genotipo G1. Las muestras argentinas también correspondían al genotipo G1, pero entre ellas se registraron algunas microvariantes de los genes mitocondriales citocromo c oxidasa-1 (CO1) y NADH deshidrogenasa- 1 (ND1). La comparación de las características de los quistes y de la morfología de los ganchos rostelares del metacestode mostró ciertas diferencias. En conclusión, existen algunas diferencias genéticas y fenotípicas entre los aislados de E. granulosus de Argentina y España. Probablemente estas diferencias, más importantes desde el punto de vista epidemiológico, podrían estar relacionadas con diferentes etapas en los programas de control de la enfermedad en los dos países. Estudios adicionales que involucren datos epidemiológicos, morfométricos y moleculares provenientes de otros tipos de ganado contribuirán a clarificar y ampliar la información aportada por este trabajo.

Japanese monkey (Macaca fuscata) with alveolar echinococcosis after treatment with albendazole for 10 years: serodiagnosis and determination of albendazole metabolites.

In 1997, an outbreak of alveolar echinococcosis in Japanese monkeys (Macaca fuscata) occurred in a zoo in Hokkaido, Japan. Twelve infected monkeys from a colony (n = 57) were diagnosed serologically by Western blotting, and ultrasonography showed the presence of tumor-like tissue in the livers of nine monkeys. The 12 infected monkeys have been treated with albendazole for 10 years without surgical resection. Ten of these monkeys have died so far; diagnoses were confirmed histopathologically through autopsy. Two of these monkeys are still alive. Recently, a significant difference between the two living monkeys was recognized. A difference in curative effect was demonstrated between the two living monkeys by radiography, contrast enhanced computed tomography, and contrast ultrasound. One showed metastasis to various organs, and the other appeared to be almost cured, as demonstrated by size reduction and calcification of the lesion after albendazole treatment for 10 years. This time, serological reexamination was performed to corroborate this apparent difference. The serological tests supported the preliminary imaging findings. In addition, the presence of albendazole metabolites in sera was confirmed by high-performance liquid chromatography. In this study, it was demonstrated that tests which have been used in human cases were also effective for diagnosing alveolar echinococcosis and for assessing curative effects in nonhuman primates such as M. fuscata.

Bacteria isolated from 25 hydatid cysts in sheep, cattle and goats.

Bacteria were isolated from 12 of 25 hydatid cysts collected from the lungs and livers of cattle, sheep and goats slaughtered in the province of Messina, Sicily, Italy. Citrobacter freundii was isolated from seven of the cysts, Aeromonas hydrophila from three, Staphylococcus species from two, Salmonella species from two and Escherichia coli and Proteus vulgaris from one.

Evaluation of nitazoxanide and oxfendazole efficacy against cystic echinococcosis in naturally infected sheep.

Cystic echinococosis (CE) is a public health problem caused by Echinococcus granulosus. We aimed to determine the efficacy of nitazoxanide (NTZ) and oxfendazole (OXF) against CE in naturally infected sheep. A total of 151 ewes were assigned to the following groups: 15 mg/kg of NTZ weekly for five weeks (NTZ5); two rounds of 15 mg/ kg of NTZ a day for five days (NTZ5x2) two weeks apart; 30 mg/kg of OXF a week for 11 weeks (OXF11); 30 mg/kg of OXF plus 15 mg/kg of NTZ a week for 11 weeks (OXF/NTZ); and the control group. OXF11 and OXF/NTZ decreased the number of fertile cysts, increased the number of degenerated cysts, and were more efficacious (49.6-61.2%) against lung cysts and liver cysts (91.8-100%) than any other treatment group. OXF might be an additional strategy for control programs and an optional treatment of human CE after it is licensed.

Echinococcus granulosus: biological comparison of cattle isolates from endemic regions of Argentina and Spain.

In the present study we have compared cattle isolates of Echinococcus granulosus from Argentina and Spain. The aim was to compare and determine if there exist phenotypic and genetic differences within E. granulosus cattle isolates between an endemic area of Spain (where the disease is mainly restricted to a sheep-dog cycle) and an endemic area of Argentina (where cattle are the most abundant intermediate hosts). The Spanish samples were previously identified as G1 genotype. The Argentinean samples were also identified as G1, but some variants were found for the cytochrome c oxidase-1 (CO1) and NADH dehydrogenase-1 (ND1) mitochondrial genes. When comparing the cyst features and the morphology of the larval rostellar hooks in both regions, some differences were found. The morphometric analyses of the larval rostellar hooks showed the existence of two distinct clearly separated groups (one corresponding to the Argentinean samples and the other to the Spanish ones). In conclusion, there are some genetic and phenotypic differences within E. granulosus cattle isolates from Argentina and Spain. Probably these differences, more important from an epidemiological point of view, are related to different steps in the disease control in both countries. Further studies involving other epidemiological, morphometric and molecular data, including other types of livestock, would contribute to clarify and expand the present work.

The important zoonoses in the protected areas of the Tatra National Park (TANAP).

The northern part of Slovakia constitutes an important tourist and recreational area of the country. Protected localities of the Tatra National Park (TANAP) are characterised by specific ecological conditions. The high numbers of animals inhabiting protected areas of the TANAP and their potential encounters with tourists contribute to a risk of transmission of important parasitic zoonoses. The aim of presently reported study was to perform a long-lasting and detailed survey on the occurrence of zoonotic agents (in particular Echinococcus multilocularis and Trichinella spp.) with particular emphasis on the interactions between the environment, the host, and the parasite as well as the adaptation mechanisms in individual ecosystems. Within the pilot part of the study, which started in 2007, in total 397 samples of wild predators representing 10 species belonging to families Canidae, Felidae, Mustelidae and Ursidae were investigated. Helminthological necropsy (modified sedimentation and counting technique) and artificial digestion method were used for the parasites detection. Multiplex PCR approach has been used for species identification of Trichinella isolates obtained from infected animals. E. multilocularis was found in 42.7% of foxes and 1 raccoon dog. Trichinella larvae were present in 16.7% of foxes, 37.9% of martens, 33.3% of polecats, 1 bear and 1 lynx. All animals were infected by T. britovi.