Prevalence of Fascioloides Magna in Galba Truncatula in the Danube Backwater Area East of Vienna, Austria

Home , Galba truncatula, Haplometra cylindracea

Wien. Tierärztl. Mschr. - Vet. Med. Austria 98 (2011), 261 - 267

From the Museum of Natural History Vienna, 3. Zoology (Invertebrates)1 and the Institute of Parasitology, Department for Pathobiology, University of Veterinary Medicine, Vienna2

Prevalence of Fascioloides magna in Galba truncatula in the Danube backwater area east of Vienna, Austria

C. HÖRWEG1, H. PROSL2, W. WILLE-PIAZZAI2, A. JOACHIM2 and H. SATTMANN1 received July 14, 2011 accepted for publication October 18, 2011

Keywords: Galba truncatula, Fascioloides magna, Fascio- wurde der Leberegel in freier Wildbahn erstmals im Jahr la hepatica, digenea, Danube backwater area, Austria. 2000 in Fischamend, südöstlich von Wien, in Rothirschen (Cervus elaphus) und ab 2001 in Rehen (Capreolus Summary capreolus) nachgewiesen. Galba truncatula, eine amphi- Fascioloides magna is a digenean parasite of various bisch lebende Schnecke aus der Familie Lymnaeidae, ist wild ruminants. It was originally introduced to Europe from in Europa der wichtigste Zwischenwirt dieses Leberegels North America in the 19th century and first recorded in the (Abb. 1). In der vorliegenden Studie wurden die Donau- wild in Austria in 2000 at Fischamend, southeast of Vien- Auen zwischen Wien und Bratislava, sowie Auen am na. Later, several cases were detected in the Danube Unterlauf der March einerseits auf das Vorkommen der backwater region between Vienna and Bratislava. The lym- Überträgerschnecken, andererseits auf die Infektion mit naeid snail Galba truncatula is known to act as natural Stadien von digenen Trematoden, insbesondere Fascioloi- intermediate host of F. magna in Europe. des magna, untersucht. In this study prevalence and distribution of the parasite Material und Methode in the snail host were investigated between August 2004 Im Zeitraum von Juli 2004 bis September 2005 wurden and September 2005. Snails were dissected, digeneans im Gebiet des Nationalpark Donau-Auen sowie bei Fischa- roughly determined by light microscopy. Species determi- mend, Hainburg, Markthof und Marchegg insgesamt 109 nation of rediae and cercariae with fasciolid appearance Standorte - zum Teil mehrmals - beprobt (Abb. 2). Gefun- was carried out by PCR and DNA sequencing. A total of dene Schnecken wurden seziert und im Lichtmikroskop 109 locations in this region were investigated. auf Stadien digener Trematoden hin untersucht. Redien Galba truncatula was recorded at 38 locations, usually und Zerkarien mit Fasciolidae-artiger Morphologie wurden with high frequency at swampy embankments of slow run- mittels molekularer Methoden bestimmt. ning water, near the water line. A total of 10,059 individu- Ergebnisse als of Galba truncatula were examined, of which 244 were Die Leberegelschnecke Galba truncatula wurde an 38 infected (prevalence of 2.43 %) with different digeneans. (von 109 untersuchten) Standorten (Abb. 2), in meist The findings included stages of F. magna, Fasciola hepati- großer Dichte, gefunden. Die Art ist im untersuchten ca, Paramphistomum sp., Haplometra cylindracea, Tylodel- Gebiet sehr verbreitet. In erster Linie wurden dichte Popu- phis sp., Notocotylus sp. and of Echinostomatidae as well. lationen an den schlammigen, flachen Ufern langsam Some stages remained unidentified. F. magna was record- fließender Gewässer gefunden. Alleine an den Standorten ed only 3 times (prevalence of 0.03 %). All findings origi- um Fischamend (Fischa) und Orth (Große und Kleine nated from one locality at the northern bank of the river Binn) konnten an die 8.000 Individuen gesammelt werden Fischa. Another finding records F. hepatica, from a locality (Tab. 1). Insgesamt wurden 10.059 Galba truncatula unter- nearby. sucht. 244 Schnecken waren mit digenen Trematoden Further monitoring of final hosts and mapping of inter- befallen, was einer Gesamtprävalenz von 2,43 % ent- mediate snail hosts are recommended to estimate the epi- spricht. Gefunden wurden Fascioloides magna, Fasciola demiology of the parasite and dispersal risk onto other hepatica, Paramphistomum sp., Haplometra cylindracea, regions and hosts. Tylodelphis sp., Notocotylus sp. und Echinostomatidae (Abb. 3). Schlüsselwörter: Galba truncatula, Fascioloides magna, Fascioloides magna wurde lediglich in Fischamend am Fasciola hepatica, Digenea, Donauauen, Österreich. Nordufer der Fischa im Sommer gefunden. Die Prävalenz von F. magna im Untersuchungsgebiet war mit 0,03 % Zusammenfassung außerordentlich niedrig. Des Weiteren gelang ein Fund Prävalenz von Fascioloides magna in Galba truncatula einer mit Redien von Fasciola hepatica, dem heimischen in den Donauauen östlich von Wien, Österreich Großen Leberegel, infestierten Schnecke - ebenfalls am Einleitung Nordufer der Fischa (Abb. 4). Der amerikanische Riesenleberegel, Fascioloides Diskussion magna, ist ein digener Trematode, der in der Leber ver- Die Daten belegen, dass ein autochthoner Zyklus des schiedener Wiederkäuer lebt. Der Parasit wurde im 19. eingeschleppten Parasiten etabliert ist. Hohe Schnecken- Jahrhundert aus Nordamerika eingeschleppt. In Österreich dichten, unmittelbar hinter dem Ufer liegende Über-

261 Wien. Tierärztl. Mschr. - Vet. Med. Austria 98 (2011) schwemmungswiesen, die dem Wild als Weiden dienen, Weiteres Monitoring des Wildes scheint angebracht, um sowie nahe gelegene Fütterungsplätze lassen diesen die Dynamik der Infektion im untersuchten Gebiet im Auge Fundort an der Fischa ideal für die Übertragung erschei- zu behalten und die Risiken einer Ausbreitung besser nen. Zwar ist die Prävalenz des Parasiten in den Überträ- abschätzen zu können. Auch eine Einschätzung der gerschnecken sehr gering, doch kann dies auf die voran- Gefährdung von Weidevieh sollte vorgenommen werden. gegangene Medikation des Wildes zurückgeführt werden. Eine Kartierung potenzieller Vektorschnecken der Familie Überdies kann mit stark schwankenden Prävalenzen auf- Lymnaeidae (mit den Genera Galba, Stagnicola, Radix grund von jährlich unterschiedlichen Umweltbedingungen, u.a.) entlang von vermuteten Ausbreitungswegen ist drin- wie Hochwasser und Temperatur, gerechnet werden. Der gend zu empfehlen. Fund des einheimischen Leberegels Fasciola hepatica im gleichen Gebiet ist von human- und veterinärmedizini- Abbreviations: BLAST = basic local alignment search tool; bp = schem Interesse. Als wichtigste unmittelbare Maßnahmen base pair; DNA = deoxyribonucleic acid; dNTP = deoxyribonuc- erscheinen die gezielte Lenkung des Wildes durch Verle- leotide triphosphate; F. hepatica = Fasciola hepatica; F. magna = gung der Fütterungsstellen an Plätze mit geringem Infekti- Fascioloides magna; G. truncatula = Galba truncatula; PCR = onsrisiko, sowie das ausreichende Lagern des Heus von polymerase chain reaction; Taq = Thermus aquaticus Überschwemmungswiesen aus Risikogebieten, geeignet.

Introduction (FRANK, 1982, 1984; WEIGAND and STADLER, 2000; RECKENDORFER and SCHÄFER, 2003). Within the snail host the parasite undergoes an enormous non-sexual Fascioloides magna, the Giant Liver Fluke, was intro- reproduction (ŠPAKULOVÁ et al., 2003). Because of the duced from North America to Europe in the 19th century. eminent impact of the snail hosts for the epidemiology, dis- Definitive hosts in America cover a wide range of ungu- persal and infection probability of the liver fluke we investi- lates including cervids and cattle (PYBUS, 2001). Records gated Galba truncatula and other snails from environments in Central Europe are restricted to cervids. The parasites close to both banks of the Danube in Austria between have a considerable pathogenicity and infection may be Vienna and Bratislava, as well as some localities at the lethal, particularly in roe deer. The European distribution lower section of the (border) river March in Austria. In total includes populations in game parks and wild environments more than 10,000 snails were investigated for the occur- in Italy, Germany, Czech Republic, Poland, Slovakia, Hun- rence of trematode stages in general and fasciolids in par- gary, Croatia and Serbia (BASSI, 1875; SALOMON, 1932; ticular. Firstly, we aimed to elucidate the distribution pat- SLUSARSKI, 1955; ERHARDOVÁ, 1961; MAJOROS and terns and seasonal dynamics of G. truncatula in the inves- SZTOJKOV, 1994; RAJSKÝ et al., 1994; MARINCULIC et tigated area. Secondly, we wanted to describe the dige- al., 2002; NOVOBILSKÝ et al., 2005; URSPRUNG et al., nean infection status of G. truncatula. Thirdly, we wanted to 2006). First evidence of F. magna in Austria was recorded highlight the epidemiology of F.magna and other fasciolids. from a game reserve (PFEIFFER, 1983) and almost 2 Finally, we wanted to estimate the impact of the parasites decades later in the wild in 2000 from hunting grounds in the environment and discuss the need of counteractions. within the Danube backwaters east of Vienna (WINKEL- MAYER and PROSL, 2001). The next parasitized populations are known from neighbouring areas in Hungary Material and methods (GICZI, 2008) and Slovakia (RAJSKÝ et al., 2002). Though the deer populations in most of these grounds are rather This study took place in the surroundings of the river small and isolated, parasite transfer must have occurred Danube between Vienna, Austria and Bratislava, Slovakia. from there. Populations from Hungary and Slovakia seem The study region includes the Nationalpark Donau-Auen to originate from already established Czech populations and adjacent areas and the angle between the Danube and in general the European populations are assumed to and the estuary of the river March (Fig. 2). In total 109 have multiple origins (KRÁLOVÁ-HROMADOVÁ et al., localities were investigated for the occurrence of Galba 2011). truncatula. Different aquatic and semiaquatic localities like The life cycle of F. magna resembles that of Fasciola river shores, backwaters, ponds, temporarily drying ditch- hepatica, the European Liver Fluke, and includes an obli- es, grooves, rainwater puddles, swamps and temporarily gatory intermediate snail host and a mammalian final host. flooded meadows were sampled, 32 of them more than Infection of the final host occurs from ingestion of meta- once. A few localities, which had been postulated as high- cercarial cysts on feeding plants, probably also from drink- er infection risk areas by RECKENDORFER and ing contaminated water (ESTEBAN et al., 1997; RONDE- SCHÄFER (2003) were sampled at least once a month. LAUD et al., 2004). The lymnaeid semiaquatic snail The study was conducted from August 2004 to September species Galba truncatula (Fig. 1) is the main intermediate 2005. Sampling was performed mainly by hand; in few cas- host of F. magna in Europe (ERHARDOVÁ-KOTRLÁ, es sieves were used. Only G. truncatula was collected in 1968), but some related species of the genera Stagnicola, higher numbers, from other aquatic snails small random Radix and Omphiscola may also represent possible inter- samples were taken. Collection times, number of collecting mediate hosts (CHROUSTOVÁ, 1979; FALTÝNKOVÁ et persons and number of collected snails were recorded in al., 2006; RONDELAUD et al., 2006). G. truncatula is by far order to calculate an estimate of the population density. the most abundant aquatic snail species in the environ- After transport to the laboratory the snails were examined ment studied, even though the distribution patterns and as soon as possible or preserved in 80 % ethanol and environmental preferences are not known in detail stored for later dissection. First, shells were measured and

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Tab. 1: Galba truncatula - samplings along the Danube backwater east of Vienna with the highest snail frequencies

DateDate Samplings Frequency*

27.6.05 Fischamend - Fischa northern bank 388 20.6.05 Fischamend - Fischa northern bank 310 1.8.05 Fischamend - Fischa northern bank 291 8.8.05 Fischamend - Fischa northern bank 192 1.8.05 Fischamend - Fischa northern bank Hirschensprung 180 8.8.05 Fischamend - Fischa northern bank Hirschensprung 176 15.9.04 Regelsbrunn - Donaulände 167 3.11.04 Orth - Große Binn 141 11.9.04 Orth - Kleine Binn 121 8.9.05 Fischamend - Fischa northern bank 118 3.11.04 Orth - Ford between Große Binn and Elenderhaufen 103 22.9.05 Maria-Ellend - Fischa southern bank 97 22.9.05 Regelsbrunn - between the backwaters 95 8.8.05 Mannswörth - Poigen Au 70 3.11.04 Orth - Kleine Binn 59 21.7.05 Fischamend - floodplain area within the town 53 27.7.05 Stopfenreuth - Roßkopfarm 51 15.9.04 Regelsbrunn - between the backwaters 49 15.6.05 Orth - Entenhaufen groove 48 8.9.05 Fischamend - Fischa northern bank Hirschensprung 48 12.10.04 Orth - Ford between Große Binn and Elenderhaufen 45 21.7.05 Fischamend - Fischa northern bank 42 27.7.05 Orth - Ford between Große Binn and Elenderhaufen 41 8.6.05 Fischamend - Fischa northern bank 40 7.7.05 Orth - Ford between Große Binn and Elenderhaufen 39 28.11.04 Schönau 39 8.8.05 Fischamend - backwater near Rostiger Anker 38 7.7.05 Orth - Große Binn 35 4.5.05 Orth - Kleine Binn 35

* frequency = number of collected snails / person / 10 minutes assigned to 4 size classes: <2, 2-5, 5-8, >8 mm. Before as part of the sample preparation kit) and DNA was investigation of the soft body, the shell was smashed and extracted according to the manufacturer’s instructions. removed. Dissection included opening the surface tissues PCR was carried out using primers within the internal and fractionizing foot, digestion gland and reproduction transcribed spacer region of the ribosomal DNA of fasci- tract. Dissections were conducted under a stereomicro- olids as described by ADLARD et al. (1993), i.e. 5‘-GGTAC- scope (MZ125) using 10 - 40 x magnification. Digeneans CGGTTGGATCACTCGGCTCGTG-3‘ (forward) and 5‘- detected in the snails were studied with a translucent TATGCTTAAGTTCAGCGGGT-3‘ (reverse) in a reaction microscope (Axioskop, using 40 - 1,000 x magnification), volume of 25 μl with 1.5 mM magnesium chloride, 200 μM identified morphologically as far as possible and docu- of dNTPs, 10 pmol of each primer, and 0.75 U of Taq poly- mented photographically. Different sources of identification merase in the corresponding buffer. Cycling conditions literature were used (GRABDA-KAZUBSKA, 1970; were: initial denaturing at 94 °C for 5 min, followed by 38 ERHARDOVÁ-KOTRLÁ, 1971; SEY, 1979; PYBUS, 2001; cycles of denaturing (92 °C, 30 s), annealing (60 °C, 30 s), JONES, 2005; ECKERT et al., 2008). and elongation (72 °C, 1 min). DNA samples extracted For further molecular identification of species, fasciolid from adult worms (F. hepatica, F. magna) served as posi- stages and young or undifferentiated rediae were frozen in tive controls. Expected product sizes ranged from 550-600 200 μl of lysis buffer (High Pure PCR Template Preparation bp. Bands of that size were sequenced (ABI Prism® Model Kit) at -18 °C for subsequent DNA extraction and sequence 310) and compared to available sequences in the Gen- determination. For molecular differentiation frozen samples Bank® database (http://www.ncbi.nlm.nih.gov/genbank/) were thawed, digested with 40 μl of proteinase K (solution using BLAST® (http://blast.ncbi.nlm.nih.gov/Blast.cgi).

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Fig. 1: Lymnaeid snail Galba truncatula in the mud near a river shore; shell height approx. 8 mm

Fig. 2: Investigation area between Vienna, Austria and Bratislava, Slovakia

circles = investigated points; red circles = findings of Galba truncatula

Source of supply 38 locations were distributed as follows: 20 on the banks of ABI Prism® Model 310 (Applied Biosystems, Vienna, backwater systems, 14 directly on river banks (Danube, Austria); Axioskop (Zeiss, Vienna, Austria); High Pure PCR Fischa, March and Stempfelbach), 2 in ditches, 1 in a pond Template Preparation Kit (Roche Diagnostics GmbH, Vien- and 1 in a wheel track. No findings were recorded in marsh na, Austria); MZ125 (Leica Microsystems, Vienna, Austria); areas and flooded meadows. Taq polymerase (Promega GmbH, Vienna, Austria) Population dynamics of G. truncatula Results Most of the snails (95 %) were in the medium size classes, only 2.3 % were smaller than 2 mm and 2.5 % larger than 8 mm in size. The seasonal size distribution showed 2 In total 10,059 Galba truncatula were collected and peaks within the study year, 1 in spring and 1 in autumn examined. The most abundant populations were found at (Fig. 3). This size distribution is in agreement with the sites in Fischamend and Orth with over collected 8,000 URSPRUNG (2005) and gives an indication of the repro- snails. Estimates of the population density were as high as duction dynamic of the snails. 200 individuals per square meter. Highest collecting frequencies were reached at the bank of river Fischa (Tab. 1). Trematode diversity in G. truncatula Potential intermediate host snails of the genus Stagni- In total 244 specimens of G. truncatula (of 10,059 col- cola were found at 5 localities, 21 specimens were exam- lected) were infected (prevalence 2.43 %) with a diverse ined. Snails of the genus Radix were found at 33 sites, in spectrum of digeneans. The findings included stages of F. total 107 specimens were examined. Fasciolids could not magna, F. hepatica, Paramphistomum sp., Haplometra be detected, neither in Stagnicola nor in Radix snails. cylindracea, Tylodelphis sp., Notocotylus sp. and of Echi- nostomatidae as well. Some stages remained unidentified Distribution of G. truncatula (Fig. 4). G. truncatula was recorded at 38 of 109 locations (Fig. 2), usually with high frequency near the water line at swampy shallow shores of slow running water courses. The

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2mm > 2 - 5mm > 5 - 8mm > 8mm 80

40 Percentage

20 Fig. 3: Seasonal size distribution of Galba truncatula; x-axis indicates month/year and y-axis percentage of distribution; colours indicate size 0 classes. 7/04 8/04 9/04 10/04 11/04 3/05 4/05 5/05 6/05 7/05 8/05 9/05

Fascioloides and Fasciola et al., 2004). Sites with high densities of G. truncatula are Only 4 findings of rediae/cercariae of fasciolid appear- often frequented by deer as drinking places. Therefore we ance were recorded by microscopic diagnosis (Fig. 5 A-C). have to consider also other watering places of deer with Molecular analysis yielded F. magna in 3 cases in summer high densities of lymnaeid snails as hot spots of infection. (June/August) at a location in Fischamend at the northern In the future, parasitological studies on fascioloids in the bank of the river Fischa and F. hepatica, the Common Liv- Danube floodplains should be extended to other lymnaeid er Fluke, once (in October) at another site at the northern snails (CHROUSTOVÁ, 1979; RONDELAUD et al., 2006). bank of the river Fischa (Fig. 6). Low prevalence of fasciolids in G. truncatula might also The obtained sequences were submitted to GenBank® be due to medication of deer in Fischamend and a subse- (accession numbers: DQ683545.1, DQ683546.1). quent decrease of egg shedding (URSPRUNG and PROSL, 2011). However, MAGE et al. (2002) showed that Discussion despite of changes in prevalence of cattle with fasciolosis, the prevalence within the intermediate host snail remained stable at a low level. Lately, investigations of snails have In the Danube floodplain forests east of Vienna, the shown an increase of infected snails 2009 in some places intermediate snail host of the liver fluke F. magna, G. trun- north of the Danube and an overall increase of G. truncat- catula, is abundant with high population densities at places ula in the studied region (HAIDER, 2010). But these fluctu- of high deer abundance. This is also confirmed by further ations might also be due to other environmental influences, studies (HAIDER, 2010; LIESINGER, 2011; URSPRUNG like the annual water dynamics in the floodplains or climat- and PROSL, 2011). Highest snail densities were found at ic deviations. For instance the infection peak of deer in gently inclined muddy embankments of slow running 2007 might be in connection with increased flood inci- water. Especially the site in Fischamend south of the dences in 2006 (URSPRUNG and PROSL, 2011). Danube, where Fascioloides were recorded in snails, was Concerning the method, mere dissection and micro- adjacent to a temporarily flooded meadow and close to a scopic inspection might be under suspicion to generate winter deer-feeding place. This is supposed to be an errors because young stages could be overlooked. Howev- appropriate environment for transmission (VODNANSKY, er, HAIDER (2010) compared microscopic inspection of 2005). Snails and/or infective stages might be distributed snails and DNA detection of fasciolids in randomly sam- easily via floods to the meadow. In summer the shallow pled snails and showed that there was no significant differ- water grows warm quickly and is supposed to support the ence between those 2 methods for detecting fasciolid parasite’s development within the snail and the emergence flukes. from the snails. This reveals optimum conditions for infec- In 1 case we detected stages of the autochthonous liv- tion of deer after the drawdown of the water and the grow- er fluke, F. hepatica. This finding was close to the Fasci- ing-up of vegetation (URSPRUNG and PROSL, 2011). oloides sample and the environmental situation is rather Moreover, new infections of snails with trematodes via similar. Since F. hepatica is also a pathogen of humans, miracidia from the eggs in deer faeces are easily imagina- this finding is of medical and epidemiological interest ble. Despite of rather high prevalences and intensities in (ECKERT et al., 2008). red deer (URSPRUNG et al., 2006; URSPRUNG and A catalogue of measures was proposed including (a) PROSL, 2011), prevalence in snails were extremely low. long-term monitoring of deer (liver, faeces) and snails, (b) Nevertheless, we assume that G. truncatula is the ade- game management (rest areas, shelter, attractive forage quate intermediate host for F. magna in the environment plants; if possible outside the flood plain), (c) hunting red studied. Radix species are also proven to be suitable inter- deer within the habitats of the intermediate snail host mediate hosts (FALTÝNKOVÁ et al., 2006). Final hosts (banishment, reduction), (d) regulation of the red deer might be infested with metacercariae of fasciolids not only stock to minimize transmission/infection, (e) mowing after by feeding on contaminated plants, but also by drinking the flood (SATTMANN and HÖRWEG, 2006; URSPRUNG contaminated water (ESTEBAN et al., 1997; RONDELAUD and PROSL, 2011).

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Fig. 5: Egg and larval stages of Fascioloides magna: egg with opened operculum and miracidium inside (A), redia with cercariae inside (B) and encysting cercaria trans- forming into a metacercaria (C); scale bar = 50 μm Fig. 4: Distribution of the digeneans (n = prevalences in %)

Fig. 6: Findings of Fascioloides magna (red triangle) and Fasciola hepatica (purple triangle) at the northern bank of the river Fischa near Fischamend

In order to observe the development in the study area scher Landesjagdverband (Hunting Corporation of Lower and to predict the success of measures to control Austria) and the Museum of Natural History Vienna. We are F. .magna, further monitoring of final hosts is desirable. Risk grateful to 2 anonymous reviewers for their constructive assessment for the dispersal to other regions and other suggestions. hosts, e.g. livestock, should be developed. Mapping of the potential vector snails, e.g. species of the genera Radix or References Stagnicola, both present in the area as well, should be pro- moted in order to evaluate the risks to introduce and estab- ADLARD, R.D., BARKER, S.C., BLAIR, D., CRIBB, T.H. (1993): lish the cycle in neighbouring regions. Comparison of the second internal transcribed spacer (ribosomal DNA) from populations and species of Fasciolidae (Dige- Acknowledgements nea). International Journal of Parasitology 23, 423-425. This study was supported by Nationalpark Donau-Auen BASSI, R. (1875): Sulla cachessia itterio-verminosa, o marciaia GmbH (Operator of the National Park), Niederösterreichi- die cervi, causata dal Distomum magnum. Il Medico Veterina-

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Christoph Hörweg, Dr. Helmut Sattmann, Burgring 7, 1010 MARINCULIC, A., DZAKULA, N., JANICKI, Z., HARDY, Z., Vienna, Austria; Univ. Prof. Dr. Heinrich Prosl, Walpurga Wille- LUCINGER, S., ZIVICNJAK, T. (2002): Appearance of American Piazzai, Univ. Prof. Dr. Anja Joachim, Veterinärplatz 1, 1210 Vien- liver fluke (Fascioloides magna Bassi, 1875) in Croatia - a case na, Austria. report. Veterinarski Arhiv 72, 319-325. e-mail: [email protected] NOVOBILSKÝ, A., HORÁCKOVÁ, E., KOUDELA, B. (2005): Current distribution of the giant liver fluke Fascioloides magna in the Czech Republic. Helminthologia 42, 181.

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