A. Radačovská, E. Bazsalovicsová and I. Králová-Hromadová
Diphyllobothriosis is a fish-borne parasitic zoonosis caused by so-called “broad tapeworms” or “fish tapeworms” of different genera of the order Diphyllobothriidea. Dibothriocephalus l atus (Linnaeus 1758), (syn. Diphyllobothrium latum), is a medically important type species of the genus, whose occurrence in various European regions is either regular, e.g. in the Alpine lakes region, or occasional and sporadic, e. g. in the Danube River region. For the latter, data on the detection of D. latus plerocercoids in the second intermediate fish host (European perch Perca fluviatilis), as well as in definitive hosts (human and dog), in which infection was directly linked to the consumption of infected fish from the Danube, were published more than 50 years ago. In order to assess the current situation, we aimed to find out whether D. latus is present in the natural environment of the Danube River. In total, 700 perch from five sampling sites in the Slovak part of the Danube River were examined. Plerocercoids were not detected in any fish examined, which leads to the conclusion that D. latus is currently not present in the studied aquatic environment.
K. Oberhauserová, E. Bazsalovicsová, I. Králová-Hromadová, P. Major and M. Reblánová
Molecular comparative analysis of eggs of four liver and stomach flukes of cervids and domestic ruminants, Fasciola hepatica, Fascioloides magna, Dicrocoelium dendriticum and Paramphistomum cervi, was performed using a new methodological approach for eggshell disintegration. Eggs of all species were crushed mechanically by the Teflon method (PTFE) without use of chemical reagents and an efficient disruption of eggshell was checked microscopically. The egg suspension was then subjected to DNA isolation and PCR amplification using species-specific primers that annealed to the internal transcribed spacer 2 (ITS2) region of ribosomal DNA. The size of PCR products of individual species corresponded well to the size of amplicons obtained from adult flukes. The results provided evidence that the Teflon method does not destroy the structure of egg DNA, thus making the procedure broadly applicable during coprological examinations. Molecular markers introduced here are particularly important for blanket screening and differentiation of morphologically hardly distinguishable F. hepatica, F. magna and P. cervi eggs.
M. Reblánová, M. Špakulová, M. Orosová, E. Bazsalovicsová and D. Rajský
The study describes a karyotype of a common parasite of cervids, the giant liver fluke, Fascioloides magna (Trematoda, Platyhelminthes). The chromosome set of F. magna comprises 11 pairs of chromosomes, all classified as subtelocentric except for the submeta-metacentric pair No. 8 and the submetacentric pair No. 10 (2n = 22, n = 1sm + 1sm-m + 9st). The first longest pair is 4.65 μm long and the length decreases continuously to the 1.92 μm length of the last pair No. 11. No distinct secondary constriction has been observed in mitotic preparations. Fluorescent DAPI-staining reveals distinct heterochromatin bands on all 11 chromosome pairs in the centromeric regions; another DAPI-positive bands are localized at the end of the long arms of chromosomes No. 5 and the last less distinct signals appear interstitially on the long arms of the pair No. 6. Synchronous meiotic divisions of 8-spermatocyte groups have been observed during spermatogenesis, similarly with a development of spermatocytes in other trematodes. In the first two stages of heterotypic spermatocyte division, 11 bivalents (n = 11) are regularly observed, confirming the diploid number of 22 elements. Furthermore, the present analysis summarises and discusses available cytogenetic data on Fasciolidae flukes suitable for future studies on taxonomy or phylogenetic interrelationships within the family.
E. Bazsalovicsová, I. Králová-Hromadová, M. Špakulová, M. Reblánová and K. Oberhauserová
The species-specific ribosomal internal transcribed spacer 2 (ITS2) markers were designed for PCR-based molecular differentiation of Fasciola hepatica, Fascioloides magna, Dicrocoelium dendriticum and Paramphistomum cervi, liver and stomach flukes of domestic and free living ruminants. Complete ITS2 sequences were obtained for D. dendriticum and P. cervi, for the later species, ITS2 structure was determined for the first time. Intraspecific variation within geographically distant populations was found to be either very low (F. hepatica; D. dendriticum) or even absent (F. magna; P. cervi). ITS2 regions with the absence of intraspecific polymorphisms but with interspecific sequence heterogeneity were applied for design of speciesspecific primers. The specificity of developed primers was tested on genomic DNA isolated from adult individuals of studied fluke species. Application of the primers is of particular value for molecular differentiation of morphologically hardly distinguishable F. hepatica, F. magna and P. cervi eggs after coprological examinations.
L. Juhásová, I. Králová-Hromadová, M. Zeleňáková, P. Blišťan and E. Bazsalovicsová
The combination of multicriteria analysis (MCA), particularly analytic hierarchy process (AHP) and geographic information system (GIS) were applied for transmission risk assessment of Fascioloides magna (Trematoda; Fasciolidae) in south-western Slovakia. Based on the details on F. magna life cycle, the following risk factors (RF) of parasite transmission were determined: intermediate (RFIH) and final hosts (RFFH) (biological factors), annual precipitation (RFAP), land use (RFLU), flooded area (RFFA), and annual mean air temperature (RFAT) (environmental factors). Two types of risk analyses were modelled: (1) potential risk analysis was focused on the determination of the potential risk of parasite transmission into novel territories (data on F. magna occurrence were excluded); (2) actual risk analysis considered also the summary data on F. magna occurrence in the model region (risk factor parasite occurrence RFPO included in the analysis). The results of the potential risk analysis provided novel distribution pattern and revealed new geographical area as the potential risk zone of F. magna occurrence. Although the actual risk analysis revealed all four risk zones of F. magna transmission (acceptable, moderate, undesirable and unacceptable), its outputs were significantly affected by the data on parasite occurrence what reduced the informative value of the actual transmission risk assessment.
I. Králová-Hromadová, E. Bazsalovicsová, S. Bokorová and V. Hanzelová
Ribosomal internal transcribed spacer 2 (ITS2) structure was studied in monozoic tapeworms Caryophyllaeus laticeps (Pallas, 1781) from freshwater bream Abramis brama (L.) and white-eyed bream Ballerus sapa (Pallas) and Caryophyllaeus brachycollis Janiszewska, 1951 from A. brama and Mediterranean barbel Barbus meridionalis (Risso). Homologous intragenomic ITS2 structure (474 bp) was determined for C. brachycollis from both fish hosts and for C. laticeps from A. brama (486 bp). Contrary to this, divergent intragenomic ITS2 copies (ITS2 paralogues) were detected in C. laticeps from B. sapa. They were mostly induced by different numbers of short repetitive motif (TA)n within the sequences, allowing their assortment into two ITS2 variants (457 and 467 bp). Current data represent first information on ITS2 structure/ITS paralogues in the caryophyllidean family Caryophyllaeidae with focus on their applicability in the molecular taxonomy of the genus Caryophyllaeus.
E. Bazsalovicsová, M. Špakulová, L’. Juhásová, Š. Miholics, D. Rajský and I. Králová-Hromadová
Fascioloidosis of wild and domestic ruminants is caused by giant liver fluke, Fascioloides magna (Trematoda; Fasciolidae). In Slovakia, the parasite is present in the Danube floodplain forests permanent focus for almost 30 years. Here we provide data on 11-year survey of F. magna acquired from 137 red deer (Cervus elaphus) hunted in the southwestern hunting grounds (districts Komárno and Dunajská Streda). Almost 47 % of all examined deer, including males, females and fawns, were infected with F. magna. During the studied period, the prevalence ranged between 33.3 % (2009) and 63.6 % (2007). Prevalence of fascioloidosis varied between sexes and age categories; while the lowest overall prevalence was detected in females (33.3 %), higher values were documented for red deer males (50.6 %) and fawns (43.3 %). A presence of giant liver fluke in studied regions of southwestern Slovakia deserves future attention and ongoing monitoring due to a possible threat of F. magna infection of domestic ruminants in overlapping regions.