The broad range of activities in the area of parasitology has resulted from the mission of the Parasitological Institute of SAS. The obtained results have a character of basic research and have greatly benefited various branches of parasitology, both from a national and international standpoint. Using progressive multidisciplinary and complex approaches, advanced laboratory and statistical methods focusing on epidemiologically the most important species and problems of the parasite transmission caused by the global climatic and ecological changes, have brought about a whole new array of original outcomes in every research direction, developed at the Institute. The contributions and new knowledge can be characterised as follows:
Research on systematics (taxonomy, phylogeny) and studies on morphology and biology of parasites of free-living animals have a long tradition at the Institute and within similarly oriented research. Owing to quality of scientific achievements in this field, the Institute continually sustains leading position in Slovakia.
The biodiversity of fish and bird helminths in three internationally important aquatic ecosystems; the Slovak part of the Tisa River, the Latorica Protected Landscape Area and Senné ponds, has been investigated. Two new species were erected based on this survey: the fish nematode Philometroides barbi n. sp. from Mediterranean barbel, and the bird tapeworm Anomotaenia barusi n. sp. from the little ringed plover. The generic diagnosis of Anomotaenia has been specified, and the species A. alata has been re-described. Furthermore, four following species were first found in the Slovak fauna: trematode Aspidogaster limacoides, thorny-headed worm Pomphorhynchus tereticollis, and tapeworms Atractolytocestus huronensis and Khawia sinensis. The two latter species belong to invasive alien helminths.
In the interdisciplinary study of pseudophyllidean fish tapeworms, original genomic, ultrastructural and biological characteristics, of significant value for systematics and phylogeny, were gathered. In four European representatives of the genus Eubothrium, the structure of ribosomal gene (5.8 rDNA), internal transcribed spacers (ITS-1 and ITS-2) and 14 enzyme systems have been specified. The specific structure of the spermatozoon and dynamics of spermiogenesis was first described in these parasites. In terms of microevolutionary characteristics, the description of a two-host life cycle, lacking coracidium larval stage, is of high importance.
The phylogenetic relationships in tapeworms of the order Caryophyllidea that constitutes one of the oldest groups of Eucestoda, have been first analyzed. Cladistic analyses based on morphological characteristics of all so far described genera have revealed essential marked discrepancies in current systematic classification of the order Caryophyllidea.
In crucial invasive ruminant parasite, Fascioloides magna (Trematoda), the sequence structure of a small subunit of the ribosomal gene (18S rDNA) and internal transcribed spacers (ITS1, ITS2) of nuclear ribosomal gene was originally determined and new molecular species-specific markers were designed.
Originally described karyotype of the fish tapeworm Nippotaenia mogurndae (= Amurotaenia perccotti) consists of 28 small metacentric chromosomes which is the highest number of chromosomes so far determined in tapeworms and the first cytogenetic information received within the order Nippotaeniidea. Karyotype of another tapeworm, Atractolytocestus huronensis, has been characterised by the triploid chromosome set (3n = 24) and aberrant spermiogenesis. Thorny-headed worms Pomphorhynchus laevis and P. tereticollis posses an identical number of chromosomes (2n = 6 + XX/X0); however, the fluorescent in situ hybridisation (FISH), applied for the first time in acanthocephalans, has shown that the loci of ribosomal genes in both species were located on different arms of the second chromosome pair. Original data were also obtained on nucleotide sequence of the telomeric DNA and the occurrence of the supernumerary B chromosomes in natural helminth populations.
The spermiogenesis and ultrastructure of sperm cells and vitellogenesis of the Cyathocephalus truncatus, Diplocotyle olrikii and Khawia armeniaca tapeworms have been for the first time studied in detail. The basic characteristics of tapeworm spermiogenesis have matched, unlike the sperm ultrastructure that differed among these species.
Of high importance for the Slovak science are especially the descriptions of the helminths new to the Slovak fauna and new invasive alien helminth species established in the ecosystems of national and international significance.
Original descriptions of new species and new helminth karyotypes, re-descriptions of higher systematic units and understanding of phylogeny of specific helminth groups represent a fundamental contribution for science on global scale. Numerous data have been acquired within a broad international co-operation with the partner institutions in the Czech Republic and other countries in the framework of the EU projects “FAUNA EUROPAEA” and “SYNTHESYS”.
Research in the field of parasitic zoonoses, diseases transmissible between animals and humans, has been focused on newly emerging and medically important diseases, such as echinococcosis, trichinellosis, toxocariasis, toxoplasmosis, and others.
The Institute has pursued activities in the fields of epidemiology, molecular characterization of zoonotic agents, immune responses of hosts, and human protection.
The long-term study on trichinellosis in red fox (Vulpes vulpes), main reservoir of Trichinella parasites, has been undertaken in Slovakia over the last years. Results revealed an increase of the parasite prevalence (12.6%) within the evaluated period, coupled with spread into new foci in western part of Slovakia, where the parasite was formerly absent. Based on spatial distribution analysed by the geographic information system (GIS), risk factors were identified. Compiled map of potential risk areas has been included into the European database on trichinellosis. Molecular identification of species circulating in the Slovak territory revealed a predominance of Trichinella britovi. For the first time in central Europe, Trichinella pseudospiralis (non-encapsulating species infecting both mammals and birds) has been recorded. This important human pathogen has implicated the outbreak in large-scale pig breeding in Eastern Slovakia. Molecular and biochemical analyses revealed the highest similarity of concerned isolate with geographical isolates of T. pseudospiralis from Scandinavian countries. The farm was located in area that is an important crossing of Pan-European bird-migration routes, and the parasite has been thereby likely introduced into our region via migrating raptorial birds.
New serological methods for early intravital diagnostics of trichinellosis indirect ELISA and sandwich ELISA, were standardised and validated. Using synthetised peptides and monoclonal antibodies, the correlation between time of seroconversion and infection dose of Trichinella spiralis larvae was observed.
In Slovakia, five human cases of alveolar echinococcosis, a serious, life-threatening parasitic zoonosis, have been so far diagnosed. All patients originated from regions with the highest prevalence of its etiological agent, Echinococcus multilocularis tapeworm, in red fox. These findings have resulted from the monitoring of its prevalence in the whole Slovak territory. The highest E. multilocularis occurrence was recorded in foxes originating from the regions bordering Poland in the north and the Czech Republic in the west. To better identify the risk areas, the spatial analysis of the parasite distribution has been performed and respective maps were compiled. Using the effective intravital methods of coproantigen detection and PCR, the causative agent of alveolar echinococcosis has been first detected in hound and shepherd dogs. These findings have confirmed the circulation of E. multilocularis in synanthropic cycle of Slovakia that considerably increases the infection risk for humans.
The factors influencing epidemiology of the tapeworm Echinococcus multilocularis, which has spread into 17 European countries over the last decade, were investigated. To assess whether any genetic attributes could account for the expansive spread of the parasite, 28 specimens from 10 central and western European countries were examined by sequence analysis of ND1, CO1, 12SrRNA, and ATP6 mitochondrial genes. Comparison with so far described M1 variant (prevalent in North America, China and Japan), and M2 variant (found sporadically in Germany), has revealed several fixed differences in studied European isolates across all studied genes. Gathered data pointed out for existence of the newly described major variant (designated as M3) circulating within Europe. Based on obtained sequences, the phylograms have been constructed, confirming the independent position of the M3 variant. Among its characteristic features are rapid spread into new areas and high risk for human infection.
Parasite-induced host immunosuppresion during alveolar echinococcosis and Mesocestoides corti infection, has resulted in the stimulation of Th2 immune response at the expense of Th1 components. It was found that anthelmintics might influence the ratio between Th1 and Th2 immune response. Administration of liposomes with incorporated albendazole has implied a three-fold prolongation of albendazole parasitostatic effect. Furthermore, liposomes have induced the double IFN-γ production in comparison to free albendazole. The possibility of elimination of parasite immunosuppressive effect on host immune response by immunomodulators was experimentally studied. The muramyl tripeptid-fosfatidyletanolamin immunomodulator, administered to mice with alveolar echinoccocosis alone and combined with albendazole, has increased the number of helper T lymphocytes, IFN-γ level and production of cytotoxic superoxide anion by macrophages. Parasitostatic effect of the treatment has persisted for three months longer than with albendazole alone.
To recognize risks of toxoplasmosis infection for humans, slaughter pigs were serologically examined. In positive animals, the DNA from the brain was isolated and the presence of virulent strain was confirmed. The amniotic fluid of pregnant women with suspected toxoplasmosis was examined by newly introduced PCR method. Anti-Toxoplasma antibodies were detected in 41.7% of women suffering from sterility, repeated miscarriages and risk pregnancies. Survey in humans with a higher risk to acquire zoonotic infection has revealed as high as 11.2% of seropositivity for larval toxocariasis. Sera of 2 721 patients bearing liver and lung cysts were examined for the presence of larval stages of E. multilocularis and E. granulosus. In patients suffering from alveolar and cystic echinococcosis, the available therapeutic procedures have been recommended in cooordination with respective hospitals. The IgG4 subclass of anti-Echinococcus antibodies was identified as suitable diagnostic marker for the control of the treatment efficacy.
The Institute has been established in leading position in terms of the research of parasitic zoonoses in Slovakia. Scientific outputs have been upgraded by implementing of novel PCR-based and immunological methods to identify causative agents of trichinellosis, echinococcosis, and toxoplasmosis. In addition, a higher efficacy of chemotherapeutics combined with immunomodulator or incorporated into carriers has been established.
Integration of the zoonotic research into the 5th Framework Programme of the European Union and the broad international co-operation in characterizing the agents of zoonoses from endemic areas throughout the world, along with the numerous stay conducted at well-recognized institutions, undoubtedly confirm the important position of the Institute in an international context. This was also demonstrated by the award received at the IX. EMOP Parasitology where research devoted to trichinellosis has been honoured.
Research on the natural foci-diseases has been focused on three areas:
1) research on genomics of infectious disease agents based on the direct identification of agents of new or newly emerging tick-borne human and animal infections;
2) research on molecular pathogenesis of Lyme borreliosis agents and on expression of outer surface antigens responsible for Borrelia pathogenicity;
3) ecological studies on Lyme borreliosis, anaplasmosis, ehrlichiosis and babesiosis agents, which include investigations of ecology of tick and reservoir animals.
Several molecular tools were standardized to directly detect agents of Lyme borreliosis (Borrelia burgdorferi sensu lato complex) in vectors, reservoir animals and patient tissue samples (skin, blood and cerebrospinal fluid). The restriction fragment length polymorphism (RFLP) method of the intergenic spacer 5S-23S was used to detect the diversity of Borrelia species in various localities within Central Europe, with six borreliae species being recorded. For studying the Borrelia heterogeneity at subspecies level, the single-strand conformation polymorphism method of the intergenic spacer (5S-23S PCR-SSCP) and PCR-RFLP of the outer surface protein (ospA) were employed. Using these methods a considerable intraspecific variability was revealed, demonstrated by 16 genotypes with different geographic distribution: Borrelia afzelii, B. garinii, B. valaisiana, B. burgdorferi s.s., B. lusitaniae and a new species, B. spielmanii, which was detected in vector for the first time. The OspA PCR-RFLP method, in which five different restriction enzyme were formerly used previously for differentiation of nine various OspA types, was further improved by adding the Xbal enzyme. This enabled us to distinguish further OspA types within the B. burgdorferi s.l. complex (OspA types 6 and 8 within B. garinii and the new species of B. spielmanii). The considerable inter- and intraspecific genetic polymorphism of borreliae was confirmed by this study. Within B. garinii all OspA types were found.
The constructed phylogenetic tree depicting relationships among genotypes of Lyme borreliosis agents from the Czech Republic and Slovakia has shown that B. burgdorferi s.l. complex represents a group of closely related species. Newly detected B. spielmanii formed a basal line of separate branch, comprising B. garinii, B. lusitaniae, B. sinica and B. turdi.
To understand the mechanism of Borrelia transmission in a host, the expression of important genes (flagellin, dbpA, bbk32, ospA, ospC and vIsE) in Borrelia afzelii and B. burgdorferi s.s. was studied on experimental model using Ixodes ricinus nymphs feeding on laboratory mice. Different expression patterns in individual proteins were observed. These proteins also play an important role in serodiagnosis of human Lyme disease. By incorporating in vivo expressed proteins (DbpA and VIsE) the increase in sensitivity of recombinant immunoblot was achieved. Additionally, the incidence of human Incidence of Lyme borreliosis in humans was analysed in relation to geographic conditions of Slovakia and vegetation cover using statistical modelling (PC software Gretl, SPSS 14.0 for Windows), that has been spatially evaluated by the geographic information system “ArcGIS”. The findings resulted in map design followed by the assessment of infection risk areas.
The original findings on an ecological cycle of B. lusitaniae involving lizard of the family Lacertidae was obtained. The impact of lizards on the ecology of this serious zoonosis has not been previously considered. These findings greatly contribute to the knowledge on ecology of Lyme borreliosis as a serious zoonosis, revealing new potential natural foci of the disease and its cycle in nature. They also highlight the significant role of the cold-blooded vertebrates in epidemiology of tick-borne zoonoses.
The SSCP based on the analysis of the fragment of 16S rDNA of the family Anaplasmataceae was optimised and standardized. This allowed the determination of Anaplasma phagocytophilum, the agent of human granulocytic ehrlichiosis, and its differentiation from non-pathogenic symbiotic bacteria of Neoehrlichia mikurensis, Wolbachia pipientis and Anaplasma-like. A. phagocytophilum was found to be the predominant species in ticks in Slovakia and was also confirmed in the blood of dogs with the clinical signs of infection. Anaplasma ovis was detected in sheep. These findings present the first reference on the species occurrence in Slovakia.
The important role of small mammals of the genera Apodemus, Microtus, Clethrionomys as reservoirs for Lyme borreliosis and anaplasmosis/ehrlichiosis in natural foci of various geographic areas of eastern Slovakia has been ascertained.
Enzootic oocurrence of canine babesiosis, so far considered as an imported infection from Mediterranean region, was evidenced by detection of the Babesia canis canis agent in dogs. with relevant clinical manifestations in Eastern Slovakia. The prevalence of the pathogen in D. reticulatus ornate cow tick has been as much as 40% in south-east Slovakia.
The above results contributed to the understanding of pathogenesis, ecology, and epidemiology (epizootology) of the agents of Lyme borreliosis, ehrlichiosis, and babesiosis in Central Europe.
The long-term research in the field of experimental pharmacology is currently primarily focused on the study of helminth resistance to antiparasitics and use of immunomodulators in order to increase the efficacy of therapy against tissue helminths. Resistance to drugs is a worldwide and serious problem. The prevalence and distribution of drug-resistant strains of nematodes in sheep in Slovakia were studied and monitored in the evaluated period. A total of 28 sheep herds from the whole territory were examined using the modern in vivo and in vitro methods. The mean prevalence rate of gastrointestinal nematodes was 83, with Ostertagia spp. and Trichostrongylus spp. being the most common pathogenic species, present in 93% and 100% of sheep herds, respectively. In addition to the repeatedly detected resistance to benzimidazole drugs (first records in early 90s of the last century), the resistance to the currently used ivermectin drug was also detected in four sheep herds.
To reduce the risk of developing the resistance, the research on targeted anthelmintic treatment in sheep has been initiated. New progressive tools to evaluate the health status have been implemented. Preliminary results indicate that these indicators correlated with the intensity of parasitic infection. It has been found out that the level of parasitic infection can by influenced by pasture procedures and breeding management.
New model host (Meriones) was used for experimental infection by the resistant strain of sheep nematode Haemonchus contortus. In the course of induced immunosuppression, the non-specific parasite has evolved into the fourth developmental stage.
The influence of selected cestode neuropeptides NPF and GNFFRFamide on the neuromuscular system of larvae of the Mesocestoides vogae tapeworm was investigated by measuring of larval motility and by determining of c-AMP level. The GNFFRamide caused an intensive larval motility along with the significant increase of the c-AMP concentration. The neuropeptide mainly acts as myostimulatory neurotransmitter through its own „G-protein-coupled receptor“. Unlike that, the NPF had weaker myoactive effect that has correlated with the lower c-AMP concentration in the larvae.
The experimental mice infection with Mesocestoides vogae larvae showed that co-administration of the drug praziquantel and the immunomodulator glucan incorporated into liposomes had significantly higher therapeutic effect than the drug administered alone. Combined treatment resulted in expression of other somatic and secretory antigens stimulating the synthesis of IgG and IgM antibodies. These results suggest that larvacidal effect of praziquantel may be significantly increased by co-administration with liposomised glucan via stimulation of the immune system.
In mice liver infected with Mesocestoide voage the expression of the genes coding for collagen type I and III was examined, along with the effect of praziquantel combined with glucan immunomodulator on their expression dynamics at the level of the information RNA.
In the field of research attributed to drug resistance and mechanisms of effect of combined treatment with anthelmintics and immunomodulators, the institution gained an exclusive position within Slovakia. Among the important achievements is validation of the new anthelmintic drug for treating domestic carnivores, which is currently developed by the Slovak company.
In the framework of international co-operation (COST, 6th FP of the EU), new detection methods of anthelmintic resistance are being developed and verified. The research results have been accepted worldwide which has been also substantiated by the fact that the respective scientific team was integrated into the 6th Framework Programme of the EU.
The research in the area of ecological parasitology is focused on recycling and hygienically safe utilisation of wastes contaminated by parasitic germs and monitoring of the impact of pollution in aquatic ecosystems
The influence of anaerobic and aerobic composting of organic wastes on survival of parasitic germs, causative agents of human and animal infections, was studied. The different waste types, potentially contaminated with parasitic and other pathogenic germs, were used for the recycling. During anaerobic composting of organic waste in mesophilic and psychrophilic temperature range with a maximum up to 35 şC decreased viability of Ascaris suum (by 21%) and Toxocara canis eggs (by 50%) has been observed. In aerobic composting in the compost channels in thermophilic temperature range, high temperatures (above 50 şC) and changes of physical and chemical parameters have implied total devitalisation of protozoa germs as early as after 24 hours and devitalisation of helminth germs after 48 hours. Total devitalisation of A. suum eggs in compost piles was observed within 6 days. Temperature, humidity, changes in physical and chemical parameters of the substrate and a suitable carbon – nitrogen ratio (C:N - 15-30:1) were determined as the key factors influencing viability of parasite eggs. Aerobic composting in thermophilic range reduces a risk of dissemination, survival and potential spread of endoparasitic germs in the environment. It was documented that resulted products of organic wastes processed by such composting can be used as hygienically sound manure material. After passing a law on biological waste recycling (with the exclusion of incineration and waste dumping) these findings will be widely applicable in local municipalities and agriculture.
Environmental contamination of public places by parasitic germs was surveyed and sources of this contamination were determined. Occurrence of T. canis eggs was recorded in 32% of children playgrounds in Košice, but only in 8% of playgrounds in Bratislava. In dog faeces predominantly T. canis eggs (in 22% of examined samples) have occurred, with the highest prevalence in the districts of Trebišov (69%) and Michalovce (68%), particularly in hound and stray dogs.
In aquatic ecosystems of protected landscape areas in Slovakia (the High Tatras mountain lakes and Morské Oko lake), concentrations of copper, zinc, cadmium, arsenic, lead, mercury, chromium and nickel have been surveyed. The concentration of heavy metals in the liver and muscles of the fish from these localities was low and corresponded to their actual content in bottom sediments. In the Slovak part of the Tisa River the content of mercury and PBC compounds in the tissue of fish and their parasites was monitored. Neither tissue mercury concentrations (0.07 – 0.08 mg/kg) nor PCB concentration exceeded the maximum permissible limits. Concentrations of PCB compounds in infected fish were three times lower than in uninfected fish. This has been repeatedly suggestive of the fact that heavily burdened hosts can reduce the content of toxic elements in an organism of their hosts.
The achieved results support the prominent position of the Institute in Slovakia within the field of ecological parasitology, in particular in the research on the influence of composting on survival of parasitic germs. The findings associated with the heavy metal accumulation in fish and parasite tissues are also original and of high importance.
Only few institutions exist in the international context where similarly designed research is being performed. Our results fit to mosaic of findings of the serious problem of contamination of public places, front and backyards by dogs‘ parasitic germs - causative agents of parasitic zoonoses. The finding of an international team that compost prepared from plant and animal production wastes can reduce the occurrence of pathogenic nematodes of plants is also of big value.
Within Slovakia, the subject of of plant nematology is being studied only at the Parasitological Institute. Its importance for plant protection and assessment of soil ecosystem is outstanding. The aim of the last four-year research was to comprehensively evaluate the nematodes of genera Globodera and Heterodera, serious pathogens of potato, cereals, hop and other crops. One of the greatest contributions to the knowledge about distribution of plant nematodes constitutes a finding of 10 species of genera Heterodera and Globodera, new to the Slovak fauna. In addition, in an extensive survey undertaken at 782 localities in Slovakia, 9 species of ectoparasitic nematodes of the Criconematidae family, new to the Slovak fauna were identified.
Studies on nematode communities of grassland ecosystem in mountain and submountain regions of Slovakia have shown that the diversity of nematode communities was influenced rather by the management practices of grasslands than by geographic, climatic and soil conditions. In total, 85 nematode species have been identified, including 20 plant parasitic nematode species, among which nematodes of the genera Helicotylenchus and Paratylenchus can be regarded as an important factor reducing the forage yield.
The occurrence of hop nematode Heterodera humuli in hop gardens has been determined for the first time in Slovakia. In terms of economy and phytopathology in hop production it is the most significant species of parasitic nematodes. The parasite occurred in all nine hop gardens examined, with the density of occurrence 10-33 cysts in 100 g of soil.
In cereals, the actual frequency of the occurrence of 56% of Heterodera avenae, for cereals production the most pathogenic cereal cyst nematode, has been observed. Compared to the results of 1977 and 1993, the distribution of this parasite has remained stable, whereas the intensity of the occurrence is lower by 50% at present.
Experimental study on ontogenesis of potato cyst nematode Globodera rostochiensis in nine potato cultivars susceptible to pathotype Ro1 has revealed that life cycle of parasite in our climatic conditions lasts from 49 to 61 days and during vegetation period only one parasite generation is completed. The susceptibility of the tested cultivars differed, and multiplication rate of G. rostochiensis varied from 1.9 to 46.4. The most resistant cultivars were those of the medium early Victoria, very early Vera and early Albina and have been recommended for cultivation in the areas where potato nematode can potentially occur.
Research on plant nematodes, carried out at the Institute, is of high importance for Slovakia due to its exclusivity. The members of research teams provide consultations for various organisations dealing with plant diseases and plant protection at national and international level, in particular in the field of quarantine parasitic nematodes.