Invasion Biology Meets Parasitology: A Case Study of Parasite Spill-Back with Egyptian Fasciola gigantica in the Invasive Snail Pseudosuccinea columella Daniel S. Grabner1*, Faten A. M. M. Mohamed2, Milen Nachev1, Eman M. H. Me´abed2, Abdel Hameed A. Sabry2, Bernd Sures1 1 Aquatic Ecology and Centre for Water and Environmental Research, University of Duisburg-Essen, Essen, Germany, 2 Parasitology Department, Faculty of Medicine, Fayoum University, Fayoum, Egypt Abstract The liver fluke Fasciola gigantica is a trematode parasite of ruminants and humans that occurs naturally in Africa and Asia. Cases of human fascioliasis, attributable at least in part to F. gigantica, are significantly increasing in the last decades. The introduced snail species Galba truncatula was already identified to be an important intermediate host for this parasite and the efficient invader Pseudosuccinea columella is another suspect in this case. Therefore, we investigated snails collected in irrigation canals in Fayoum governorate in Egypt for prevalence of trematodes with focus on P. columella and its role for the transmission of F. gigantica. Species were identified morphologically and by partial sequencing of the cytochrome oxidase subunit I gene (COI). Among all 689 snails found at the 21 sampling sites, P. columella was the most abundant snail with 296 individuals (42.96%) and it was also the most dominant species at 10 sites. It was not found at 8 sites. Molecular detection by PCR and sequencing of the ITS1-5.8S-ITS2 region of the ribosomal DNA (rDNA) revealed infections with F. gigantica (3.38%), Echinostoma caproni (2.36%) and another echinostome (7.09%) that could not be identified further according to its sequence. No dependency of snail size and trematode infection was found. Both high abundance of P. columella in the Fayoum irrigation system and common infection with F. gigantica might be a case of parasite spill-back (increased prevalence in local final hosts due to highly susceptible introduced intermediate host species) from the introduced P. columella to the human population, explaining at least partly the observed increase of reported fascioliasis-cases in Egypt. Eichhornia crassipes, the invasive water hyacinth, which covers huge areas of the irrigation canals, offers safe refuges for the amphibious P. columella during molluscicide application. As a consequence, this snail dominates snail communities and efficiently transmits F. gigantica. Citation: Grabner DS, Mohamed FAMM, Nachev M, Me´abed EMH, Sabry AHA, et al. (2014) Invasion Biology Meets Parasitology: A Case Study of Parasite Spill-Back with Egyptian Fasciola gigantica in the Invasive Snail Pseudosuccinea columella. PLoS ONE 9(2): e88537. doi:10.1371/journal.pone.0088537 Editor: Shoba Ranganathan, Macquarie University, Australia Received October 22, 2013; Accepted January 6, 2014; Published February 11, 2014 Copyright: ß 2014 Grabner et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was conducted in the framework of the Integrated Water Technologies (IWaTec) joint project between the Centre for Water and Environmental Research (ZWU) at the University of Duisburg-Essen and the Fayoum University in Egypt. IWaTec is funded by the German Academic Exchange Service (DAAD) and financed by the Federal Foreign Office. This publication has been supported by the Open Access program of the German Research Foundation (Deutsche Forschungsgemeinschaft). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected] Introduction technique in Fayoum area. Especially in Egypt, fascioliasis is an increasing problem, reaching prevalences in animals of sometimes The liver fluke Fasciola gigantica is a trematode parasite native to more than 50% and up to 19% in humans [5–7]. According to Africa and Asia and infects ruminants, but also humans as final estimations of the World Health Organization [8], at least 830,000 hosts. Adult F. gigantica live in the bile ducts of the liver, where they people are infected with either the introduced Fasciola hepatica or F. can reach a length of up to 76 mm. Infection with this parasite can gigantica in the Nile delta. Presence of both of these closely related cause severe disease symptoms referred to as fascioliasis [1]. species has been confirmed for Egypt [1,9,10], but they are usually Intermediate hosts are various lymnaeid snails [2], inside of which not distinguished diagnostically [11]. the parasite proliferates asexually and produces free swimming The natural first intermediate host of F. gigantica in Egypt is the cercariae that will attach to submerged surfaces, mostly plants. snail Radix natalensis, but the trematode was also found commonly These cercariae develop into encysted and durable metacercariae in the introduced species Galba truncatula [12]. Additionally, single that are transmitted when the final host ingests the metacercariae cases of F. gigantica infections were reported from Biomphalaria together with plants, or by consumption of water contaminated alexandrina and Pseudosuccinea columella in Egypt [13–15]. For the with metacercariae [3]. Due to the durability of the metacercariae, latter species, release of F. gigantica cercariae was proven in transmission can also be mediated by ingestion of terrestrial plants laboratory infections [16]. The highly invasive lymnaeid snail P. and crops that were submerged in water containing infected snails columella was introduced from North America to many countries for a couple of weeks [1,4], which is a common irrigation worldwide and was reported from Africa for the first time in the PLOS ONE | www.plosone.org 1 February 2014 | Volume 9 | Issue 2 | e88537 Fasciola gigantica in Pseudosuccinea columella middle of the 20th century, where it is now widely distributed [17– ITS1-segment of both F. gigantica and F. hepatica, to distinguish 20]. P. columella is well known as a suitable host for F. hepatica [12], Fasciola spp. from host and infections with other trematodes. but its importance for the maintenance of the natural life cycle of Sequences and additional information for primers designed for the F. gigantica and the transmission to humans is uncertain. present study are given in table 1. One 20 ml PCR reaction mix According to the parasite spill back hypothesis, invasive species contained 4 mlof56Crimson Taq buffer (New England Biolabs), are colonized by local parasites. If the invader becomes abundant 0.2 mM dNTP mix (New England Biolabs), 0.5 mM of each and the parasite can develop successfully, a high number of primer 0.5 U Crimson Taq (New England Biolabs) and 1 ml transmitting stages will develop and increase local parasite template DNA. The mix was topped up to 20 ml with PCR grade abundance and prevalence [21]. In this way, free living invasive water. The DNA was amplified by a Labcycler (SensoQuest) species may help native parasites to increase their population size under the following conditions: initial denaturation at 95uC for and extend their distribution range [22]. P. columella is a very 5 min, 40 cycles of 95uC for 30 s, annealing (temperatures see efficient invader and became an important snail host for F. hepatica table 1) for 30 s and elongation at 72uC for 30 s followed by a final in many countries [12]. We hypothesize that this might be the case elongation of 72uC for 5 min. for the P. columella/F. gigantica–system as well. As a result, infected Sensitivity of the Trem2 F/Trem1 R primer pair was lower and P. columella might be responsible for the observed increase in not all samples were amplified successfully with the Crimson Taq, infection intensity and prevalence in livestock and in the human although tested positive before with Trem1 F/Trem1 R primers. population in Egypt. Therefore, we investigated the trematode Therefore, the more robust Phire Animal Tissue Direct PCR Kit species occurring in the invasive snail P. columella, collected from (Thermo Scientific) was used for amplification with those primers. irrigation channels in the Fayoum governorate where cases of Reactions contained 10 mlof26 Phire PCR Buffer, 0.5 mMof fascioliasis are commonly reported [23,24]. We assessed the each primer, 0.4 ml Phire Hot Start II DNA Polymerase and 1 ml potential of this invasive species as a host for trematodes, especially DNA. Water was added to 20 ml. PCR conditions were 98uC for for Fasciola spp. to estimate the effect of P. columella for the spread 5 min, 40 cycles of 98uC for 10 s, annealing at 54uC for 10 s, of fascioliasis in the area and found evidence for a possible spill- elongation at 72uC for 20 s and a final elongation at 72uC for back effect on animals and the human population. 1 min. PCR conditions for the LCO1490/HC02198 primers were as described in Folmer et al. [25]. PCR products were checked by Materials and Methods standard agarose gel electrophoresis, purified with a JETQUICK PCR Product Purification Spin Kit (Genomed) according to Snail sampling manufacturer’s instructions and sent for sequencing (GATC). Snails were collected from July to September 2012, 2 month Sequences were checked for homology with database entries by after the last molluscicide treatment, from water plants (mainly BLAST searches (http://blast.ncbi.nlm.nih.gov/Blast.cgi). water hyacinths Eichhornia crassipes) and with dip nets in irrigation channels at 21 different sites in Fayoum governorate (surrounding Statistics Markaz El-Fayoum, Itsa, and Ibshway cities), Egypt. Samples were Mean prevalences and confidence intervals of parasite infections taken with permission of the local farmers owning the land were calculated with the program Quantitative Parasitology v.3.0 adjacent to the irrigation channels. No endangered or protected [26].