Highland cattle and Radix labiata, the hosts of Fascioloides magna

Background Fascioloides magna is a pathogenic fluke introduced to Europe ca 140 years ago. As it is spreading over the continent, new intermediate and definitive hosts might be involved in transmission of the parasite. In Europe, several studies reported potential new intermediate snail hosts (Radix spp.) for F. magna, and also several cases of fascioloidosis of wild and domestic animals were published. However, the data based on molecular and histological analyses confirming these findings remained unreported. This study aims to refer to unique findings of F. magna in European snails and domestic animals (the first observation in the Czech Republic in the last 30 years) and demonstrate the use of molecular techniques in determination of F. magna. Results Two snails of R. labiata naturally infected with F. magna were found; mature cercariae and daughter rediae were observed. Maturity of cercariae was checked by histological methods, however, their ability to encyst was not confirmed. Co-infection of F. magna and Fasciola hepatica in the liver of two highland cattle bulls was proved. Adult fasciolid flukes producing eggs were found in the liver pseudocysts (F. magna) and the bile ducts (F. hepatica). Identification of intermediate hosts, intramolluscan stages, adult flukes and eggs was performed by sequencing the ITS2 region. Connection of F. magna pseudocysts with the gut (via the bile ducts) was not confirmed by means of histological and coprological examinations. Conclusions For the first time, Radix labiata was confirmed as the snail host for F. magna under natural conditions and, together with the finding of F. magna infection in cattle, we can expect further transmission of F. magna from wildlife to livestock in localities shared by these hosts.


Background
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][4][5][6][7][8][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][11][12][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 -"nonspecific 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][26][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.

Ethics statement
The animals involved in the study were slaughtered in accordance with the regulations and recommendations of the Ministry of Agriculture of the Czech Republic and European Committee (The Veterinary Act 166/1999, EC regulation n. 1099/2009). The samples of the livers were examined by veterinary inspection and with the agreement of the farmers were forwarded to the team members at the Department of Parasitology, Faculty of Science, Charles University in Prague.

Intermediate host (snail) sampling and examination
In June 2011 and July 2012, Radix spp. snails were collected at a locality with previous findings of F. magna -Sedliště game reserve [16] (GPS: 49°32′30.822″N 13°3 8′39.491″E). In addition, G. truncatula snails were sampled at the same locality for estimating the prevalence of F. magna in the snail population (see Table 1 for the number of collected snails).
Snails were examined by dissection under stereomicroscope. Infected snails were washed and, together with cercariae and rediae, they were preserved in 96% ethanol for further molecular determination. In addition, hepatopancreases of infected snails were fixed in Bouin's solution (Sigma-Aldrich) for further histological processing.
The species of snails were predominantly determined by molecular methods (see below). Morphological determination was based on morphometry/morphology of the shell and reproduction organs such as bursa copulatrix and its duct [15,29]. As morphological characters of reproduction organs can hardly be observed in infected individuals due to their destruction by larval stages of trematodes [30], only uninfected individuals of Radix spp. from the same locality were chosen for morphological determination.

Definitive host (cattle) sampling and examination
Adult flukes of F. magna and F. hepatica were obtained from dissected livers of naturally infected highland cattle from South Bohemia (Czech Republic). The worms were preserved in 96% ethanol and Bouin's solution for molecular and histological processing, respectively. Some living individuals were incubated in RPMI 1640 medium (Sigma) at 37°C overnight to obtain freshly laid eggs.
In addition, coprological examination using the cattle faeces sampled directly from the gut content was performed. After sedimentation/decantation of fecal samples [31], the obtained sediment was examined under microscope and the egg-positive sediment samples were preserved in 96% ethanol for further molecular determination.

Molecular determination Snails
DNA was isolated from mechanically homogenized tissue using QIAamp® DNA Mini Kit, (QIAGEN). DNA concentration was measured by spectrophotometer ND-1000 (NanoDrop®) and then the samples were stored at -20°C. PCR was carried out in total volume of 25 μl using PPP Master Mix (Top-Bio), 50 ng of snail template DNA and 1 μl of 10 μM forward primer 5′ -TGT GTC GAT GAA GAA CGC AG -3′ and reverse primer 5′ -TTC TAT GCT TAA ATT CAG GGG -3′ specific for 502 bp ITS2 region [32].

Eggs
DNA was isolated using QIAamp® DNA Stool Mini Kit (QIAGEN) from F. magna eggs obtained after incubation of living worms, or from sediments of fecal samples. The eggs were homogenized in Bead-Beater (BioSpec) [34]. The same primers and procedure as for the adults were used (see above).
All the obtained sequences were adjusted using DNA Star-Lasergene Core Suite software tool and compared with NCBI database using BLAST (Basic Local Alignment Search Tool).

Definitive hosts
After macroscopic examination of the host liver, the tissue samples with pathological changes were fixed in 4% buffered formaldehyde (for hematoxylin-eosin staining) or Bouin's solution (for Gomori trichrome staining) and embedded in paraffin according to standard procedures. Sections (5 μm thick) were stained and examined under microscope (Olympus BX50).

Examination of snails and determination of F. magna larval stages
In total, 1155 snails of the genus Radix were examined for fasciolid larval stages; two snails were infected and the prevalence was 0.18% (Table 1). Fifty individuals of G. truncatula from the same locality showed 64% prevalence. Molecular identification of rediae and mature cercariae showed that in both types of hosts the larvae belong to F. magna.

Histology of F. magna larval stages and snails
Histological evaluation of the infected snail hepatopancreas of R. labiata revealed daughter rediae with developing cercariae, and also free mature cercariae covered by a thick layer of tegument and possessing cystogenous glands full of products (Figure 2A).

Examination of definitive hosts and determination of F. magna/F. hepatica adults/eggs
In January and September 2012 four three-years old bulls of the highland cattle were slaughtered in the slaughterhouse in South Bohemia. The dissected livers exhibited pathological changes showing the infection by fasciolid flukes -F. hepatica and F. magna (Figures 3, 4 and Table 2). The infected animals were gaunt and in bad physical condition.

Fasciola hepatica
All adult worms were found in the bile ducts with thickened and calcified walls. Beige mature flukes had maximum size of ca 3 cm and leaf-shaped body with typical oral cone and "shoulders" (Figure 3). After overnight incubation (37°C) in RPMI 1640, thousands of eggs were found and collected. Miracidia hatched after 16 days of incubation (25°C). Sequential analyses confirmed determination of both F. hepatica adults in the bile ducts and the eggs of F. hepatica in the faeces.

Fascioloides magna
Surface of the infected liver exhibited dark pigmentation associated with migration of juvenile flukes (the same dark lesions were recognized also in the case of F. hepatica infection). All individuals were localized in liver pseudocysts filled by a dark suspension of pigment, metabolites and eggs. The flukes had oval shape and reddish brown color. Length of the worms ranged from 3 cm to 5 cm. Pseudocysts with dead and particularly digested individuals were also observed. During overnight incubation (37°C) in RPMI 1640, the isolated mature living flukes produced eggs (several times less than F. hepatica); however, these eggs appeared unable to develop -after 16 days of incubation at 25°C no miracidia were observed. Sequential analyses confirmed the adult worms as F. magna. No eggs of F. magna were found in faeces.

Discussion
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][43][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.

Conclusion
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.
Endnotes 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]. b Metacercariae 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.