Fascioloides magna (Bassi, 1875) (Fasciolidae; Digenea; Trematoda) is a large visceral parasite of ruminants originating from North America. Due to the international wild animal trade, F. magna was introduced to Europe in the later half of the 19^th century, probably with the white-tailed deer (Odocoileus virginianus) [1] and wapiti (Cervus elaphus canadensis) [2]; the parasite adapted to the local hosts (e.g. red deer, fallow deer) and started to spread. Recent reports from at least six countries in central Europe can be found [3–9].

Like some other digenean trematodes, the F. magna life cycle includes two hosts, aquatic snails as intermediate hosts and ruminants as definitive hosts. In North America as the place of F. magna origin, at least five snail species of the family Lymnaeidae producing cercariae being able to form metacercariae were confirmed as the intermediate hosts [10–13]. In Europe only two naturally infected species within the family Lymnaeidae were found – Galba truncatula[1, 3, 14] and Radix peregra (in the light of recent discoveries, the latter species was probably Radix labiata) [15, 16]. Only G. truncatula was shown to serve as the natural intermediate host emitting cercariae [1]. Some other lymnaeid snails distributed in the Czech Republic were tested under laboratory conditions as potential intermediate hosts of F. magna. Chroustová et al. [17] obtained cercariae (and subsequently infective metacercariae) from experimentally infected Stagnicola palustris. Erhardová-Kotrlá [1] successfully infected Radix peregra peregra and R. peregra ovata (according to the current nomenclature probably Radix labiata and Radix balthica) [15, 18], nevertheless, only mother rediae were observed. Experimentally infected Lymnaea stagnalis was able to produce a low number of mature cercariae. This was in contrast to a massive exposure of L. stagnalis to miracidia and, therefore, L. stagnalis was considered to be a less susceptible intermediate host of F. magna[19]. Omphiscola glabra and Pseudosuccinea columella can also serve as the intermediate hosts of F. magna[20]. Concerning the latter two snails, there is no report confirming the occurrence of O. glabra in the Czech Republic, but P. columella was observed in several biotopes [21]. Since P. columella is a common snail in European greenhouses adapting to the central European climatic conditions [22], potential risk of F. magna transmission via this snail can not be excluded [20].

A wide spectrum of mammals can be infected with F. magna; these animals can generally be divided into three groups with respect to compatibility of the host and the parasite, and pathological impact of the fluke [11, 23, 24]. Successful completion of the F. magna life cycle with eggs released via bile ducts to the host intestine is enabled in “specific definitive hosts” such as red deer (Cervus elaphus), fallow deer (Dama dama) and roe deer (Capreolus capreolus) [1, 11]. Infection with F. magna is tolerated by red deer and fallow deer which can survive, usually with hardly distinguishable symptoms, severe infection by hundreds of flukes. On the other hand, for roe deer the infection with a few flukes (less than 10 individuals) can be lethal [1]. The second category – “non-specific definitive hosts (dead-end hosts)” - is represented e.g. by moose (Alces alces), lama (Lama glama) and cattle (Bos taurus); these animals only exceptionally contribute to spreading of F. magna; the adult flukes can produce eggs, but these are trapped in liver pseudocysts not connected to the bile duct system [25–27]. Pathological impact on the “dead-end hosts” can differ. Whereas Foreyt and Todd [23] and Conboy and Stromberg [28] did not observe any significant health complications in infected animals, Chroustová et al. [17] recorded a reduced weight gain of infected animals (150 g/day). The third category comprises “non-typical hosts (aberrant hosts)” which are sensitive to F. magna infection, e.g., domestic sheep (Ovis aries) and domestic goat (Capra hircus) [24, 28]; the immature flukes migrate continuously throughout the host body, cause serious damage to various organs, and finally they attack the liver which often leads to death [11, 23].

In this paper two unique field findings of F. magna are described: larval stages of F. magna in the intermediate host R. labiata, and adult worms in the “non-specific definitive host” Bos primigenius f. taurus (highland cattle); the latter case is the first report of fascioloidosis in the Czech Republic in the last 30 years. Moreover, we refer to a unique case of co-infection of the same individual definitive host by F. magna and F. hepatica.

Two Radix sp. snails naturally infected with fasciolid flukes were found at the locality Sedliště (south Bohemia – Czech Republic), and determined as R. labiata according to the sequence of ITS2 region; daughter rediae and mature cercariae were observed in the snail bodies. This is the first report on Radix sp. snails naturally infected with F. magna when morphological as well as molecular identifications have been performed. Faltýnková et al. [16] referred to a natural infection of R. peregra^a with F. magna, but the snail and the parasite determination was based on morphological characters only, and molecular confirmation was missing. Faltýnková et al. [16] collected the snails at the same locality (Sedliště game reserve) and, therefore, it is likely those snails were also R. labiata as investigated in our study. Moreover, the locality represents a typical biotope for R. labiata[35, 36]. As the snails of the genus Radix occurring at the same locality can be infected with F. magna or F. hepatica[16, 37], and their intramolluscan stages are morphologically indistinguishable, molecular determination needs to be performed to reach correct results [38]. The snails harboring both flukes were not found at the locality Sedliště. Recently, Huňová et al. [29] performed the first successful experimental infection of R. labiata with F. magna, followed by molecular identification of the snail and the parasite.

The prevalence of F. magna-infected R. labiata in Sedliště was 0.18% (similarly low prevalence from the same locality has been reported by Faltýnková et al. [16]), and the prevalence of infected G. truncatula snails was 64% (100% in 2011); such prevalence rate is quite unusual (e.g. [14]) and it indicates a high contamination of the locality by F. magna eggs.

Histological evaluation of hepatopancreas from naturally infected snails revealed freely moving cercariae with released cystogenous gland products on their surface (Figure 2). This indicates incorporation of a preparatory phase before encystation of cercariae in the outer environment [39]. However, no cercariae were able to encyst, probably because they were obtained artificially by dissection. Similar results were obtained by Erhardová-Kotrlá [1], Faltýnková et al. [16] and Huňová et al. [29] in experimental infections of R. labiata with F. magna^b.

Two species of fasciolid flukes were found in the livers of two bulls of highland cattle; a typical localization of the flukes - bile ducts (F. hepatica) and fibrous pseudocysts (F. magna) - was observed. Morphological characters served for parasite determination, and identity of both species was subsequently verified by molecular methods. In the past, only one report on co-localized fasciolid flukes in one liver of cattle was published [25]. Coprological examination performed in our study revealed a high number of fasciolid eggs which were indistinguishable according to their morphology [1, 40]. By applying molecular methods only the eggs of F. hepatica were determined. This finding corresponds to the literary data on pseudocysts that in the case of “non-specific definitive hosts (dead-end host)” are not connected via the bile ducts with the host gut, and the eggs accumulate in pseudocysts [23, 28]. However, some exceptions may occur, e.g., Erhardová-Kotrlá [1] experimentally infected cattle and recorded eggs in the faecal samples, although in a low number. Balbo et al [9] and Foreyt and Todd [41] noticed a low number of eggs of F. magna in the faeces of naturally infected cattle. Foreyt and Todd [41] believed that the eggs may appear due to rupture of pseudocyst. Absence of eggs in the faeces of “non-specific definitive hosts (dead-end host)” complicates intravital diagnosis: it is impossible to apply routine coprological examination and, therefore, serological or other techniques should be used as an alternative.

Pathological impact of F. magna infection on cattle is questionable. Conboy and Stromberg [28] focused on analysis of blood samples. In addition, liver enzyme activities were monitored. These enzymes are probably a sensitive marker of liver damage by F. hepatica[42–44], however, in case of F. magna no significant difference between infected and uninfected individuals was recorded. Moreover, the infection (contrary to F. hepatica infection) did not affect the weight gain [45]. Besides cattle, also guinea pigs served for experimental infections. Although all the infected animals died and the pathological impact was evident, most of the monitored parameters were comparable to the control [28]. This implies that fascioloidosis is a serious disease with hardly observable diagnostic markers. In our study both bulls were in bad condition (e.g., chronic cachexy was mentioned in the veterinary report). We can speculate that it was probably due to F. hepatica infection, because in the liver of each bull at least 130 F. hepatica adult worms were found, whereas the number of F. magna adults was up to 10 individual flukes.

The infected bulls came from a South Bohemian biofarm with restricted therapeutic intervention and ideal natural condition for transmission of F. magna, e.g., wet pastures, natural streamlet and no effective fences. Erhardová-Kotrlá [1] carried out detailed research in the same locality and marked this locality as F. magna free after 6 cattle postmortem examinations and 282 examinations of the red deer faecal samples. Therefore, our results indicate that F. magna can spread in the Czech Republic.

Two unique records of snail intermediate hosts (R. labiata) and mammalian definitive hosts (bulls of highland cattle) naturally infected with F. magna are reported in our study. Molecular techniques were used to confirm host/parasite determination; on this basis the adults of F. magna were discriminated from F. hepatica presented in the same liver. In addition, species identity of the morphologically uniform fasciolid eggs was proved. The prevalence of F. magna in R. labiata was comparable to some findings of F. magna in G. truncatula and, therefore, R. labiata might represent a potential vector of F. magna in localities ecologically unsuitable for G. truncatula (e.g. localities with acid soils). Ability of the parasite to be transmitted from wildlife to livestock represents a serious risk, especially for farmers breeding the animals less tolerant to F. magna infection (e.g., goats and sheep), and those animals whose are for some reason (e.g. ecological farming) under restrictions in terms of anthelmintic treatment. Therefore, the danger of F. magna transmission should be taken into account in farm management.

^a R. peregra is the taxonomic name which has generally been used by Czech malacologists. According to the results of generally accepted phylogenic analyses, R. peregra is synonymous with R. labiata which represents a valid taxon. In Western Europe the name of R. peregra was also used, but for different species currently named as R. balthica, see Schniebs et al. [15, 18].

^bMetacercariae of F. magna were found on the wall of Petri dish after dissection of experimentally infected R. labiata (Pankrác 2013, unpublished), but experimental infection of a definitive host was not performed.