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A multiplex PCR for the detection of Fasciola hepatica in the intermediate snail host Galba cubensis Annia Alba a , Antonio A. Vázquez b , Hilda Hernández a , Jorge Sánchez b , Ricardo Marcet a , Mabel Figueredo a , Jorge Sarracent a , Jorge Fraga c,∗ a Laboratorio de Anticuerpos Monoclonales, Departamento de Parasitología, Instituto de Medicina Tropical “Pedro Kourí”, Ave Novia del Mediodía km 6 ½, AP 601, La Habana, Cuba b Laboratorio de Malacología, Departamento de Control de Vectores, Instituto de Medicina Tropical “Pedro Kourí”, Ave Novia del Mediodía km 6 ½, AP 601, La Habana, Cuba c Laboratorio de Biología Molecular, Departamento de Parasitología, Instituto de Medicina Tropical “Pedro Kourí”, Ave Novia del Mediodía km 6 ½, AP 601, La Habana, Cuba
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Article history: Received 23 October 2014 Received in revised form 1 May 2015 Accepted 9 May 2015 Keywords: Fasciola hepatica Galba cubensis Multiplex PCR Epidemiology Diagnosis
a b s t r a c t Fasciolosis is a snail-borne trematode infection that has re-emerged as a human disease, and is considered a significant problem for veterinary medicine worldwide. The evaluation of the transmission risk of fasciolosis as well as the efficacy of the strategies for its control could be carried out through epidemiological surveillance of the snails that act as intermediate hosts of the parasites. The present study aimed to develop the first multiplex PCR to detect Fasciola hepatica in Galba cubensis, an important intermediate host of the parasite in the Americas and especially in the Caribbean basin. The multiplex PCR was optimized for the amplification of a 340 bp fragment of the second internal transcribed spacer (ITS-2) of F. hepatica rDNA, while another set of primers was designed and used to amplify a conserved segment of the nuclear 18S rDNA of the snail (451 bp), as an internal control of the reaction. The assay was able to detect up to 100 pg of the parasite even at high concentrations of snail DNA, an analytical sensitivity that allows the detection of less than a single miracidium, which is the minimal biological infestation unit. A controlled laboratory-reared G. cubensis – F. hepatica system was used for the evaluation of the developed multiplex PCR, and 100% sensitivity and specificity was achieved. This assay constitutes a novel, useful and suitable technique for the survey of fasciolosis transmission through one of the main intermediate hosts in the Western hemisphere. © 2015 Elsevier B.V. All rights reserved.
1. Introduction The liver fluke Fasciola hepatica is a globally distributed trematode and the main causative agent of fasciolosis (Mas-Coma et al., 2009). The parasite is characterized by a complex life cycle that includes switching through several larval stages and the development inside two very different hosts. Adults of F. hepatica develop inside mammals where they can live for years feeding on the host and laying thousands of eggs per day (Andrews, 1999). A variety of domestic mammals (e.g., sheep, cattle, buffaloes) can act as definitive hosts of F. hepatica and in fact, high prevalence of the parasite in these animals is reported worldwide (Mas-Coma et al., 2009). Consequently, fasciolosis is a serious animal health problem that
∗ Corresponding author. Tel.: +53 7 255 3112; fax: +53 7 204 6051. E-mail address: [email protected] (J. Fraga).
causes significant economic losses in several developed and developing countries (Kaplan, 2001; Espinoza et al., 2010). Humans can be affected by this parasite as well, acting as definitive hosts, and in a number of countries the disease is a significant problem for public health (Mas-Coma et al., 2009). The development of several larval stages of the parasite occurs inside freshwater snails of the family Lymnaeidae, which act as intermediate hosts. Hence, if snails occurring in the field (e.g., grazing pastures and flooded crops such as watercress and rice fields) are discovered infected with F. hepatica, direct control strategies aimed to reduce their populations or avoid contact with definitive hosts will mostly control transmission. The surveillance of high risks areas where intermediate and definitive hosts occur could help to prevent or control potential outbreaks. There are many lymnaeid species known as intermediate hosts of fasciolosis worldwide (Correa et al., 2011). The transmission of the disease is dependent on the lymnaeid hosts that occur in a par-
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Please cite this article in press as: Alba, A., et al., A multiplex PCR for the detection of Fasciola hepatica in the intermediate snail host Galba cubensis. Vet. Parasitol. (2015), http://dx.doi.org/10.1016/j.vetpar.2015.05.012
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ticular geographical area and their roles regarding transmission is influenced by the local adaptation of the species (Dar et al., 2013; Vázquez et al., 2014). In the Caribbean area, the lymnaeid snail Galba cubensis (formerly known as Lymnaea (Fossaria) cubensis) is considered the main intermediate host of F. hepatica (Gutiérrez et al., 2011). However, its presence in some regions of North (Kaplan et al., 1997) and South America (Bargues et al., 2011; Medeiros et al., 2014), where it co-exist with other snail hosts complicates the epidemiological scenario of fasciolosis. Several investigations have been conducted, mainly in Cuba where fasciolosis is endemic in livestock, regarding the role of G. cubensis in F. hepatica trans˜ mission (Vázquez et al., 2014), its ecology and distribution (Canete et al., 2004; Vázquez et al., 2009), and control strategies for its management (Perera et al., 1991). A DNA probe assay was developed and validated in field-collected G. cubensis (Kaplan et al., 1997) but the complicated and time-consuming technique of DNA hybridization could limit its application during large-scale surveys of F. hepatica infection in snails. Moreover, several PCR-based methods have been developed to detect F. hepatica infection in snail hosts (i.e., Magalhães et al., 2004; Cucher et al., 2006; Caron et al., 2011) but to our knowledge there are no studies reporting these PCR techniques on G. cubensis. So far, the detection of the parasite in this particular intermediate host mainly relies on the classical parasitological examination of field-collected snails. The aim of this study was to develop the first multiplex PCR to detect F. hepatica-infected G. cubensis. A previously reported set of primers was used to amplify a fragment of the second internal transcribed spacer (ITS-2) within the F. hepatica ribosomal DNA (rDNA). In addition, a novel set of primers was designed to amplify a conserved segment of the 18S rDNA of G. cubensis and other snails from the Lymnaeidae taxon, in order to use it in the assay as an internal control. The method was evaluated on samples of experimental snail-parasite system using laboratory-reared G. cubensis populations.
2. Material and methods 2.1. Parasites and snails Adults of F. hepatica were collected from the livers of infected cattle and placed in saline solution (0.85% NaCl) supplemented with 5% glucose for transport and egg laying. Identification of recovered flukes was done following Jones (2005). Eggs were then thoroughly washed and preserved in saline solution with Gentamicin, at 4 ◦ C until use. Adults of F. hepatica were preserved in 95% ethanol. G. cubensis was reared in the Laboratory of Malacology of the Institute of Tropical Medicine “Pedro Kourí” according to the methodology of Sánchez et al. (1995). Subsequent generations, considered free of infection, were used as negative controls. Some of the laboratory-produced snails were also used in experimental infection with F. hepatica miracidia to obtain positive control samples.
2.2. Experimental infection of G. cubensis F. hepatica eggs (see Section 2.1) were placed in distilled water and were incubated at 28 ◦ C, for 15 days in the dark. Miracidia were obtained after egg hatching induced by light exposure and experimental infection of 50 laboratory-reared G. cubensis was conducted as described by Vázquez et al. (2014). Snails were analyzed for intramolluscan larvae of F. hepatica by parasitological examination of dissected individuals (Caron et al., 2008) starting from day 14 post-exposure. Only the infected snails were preserved in 95% ethanol as positive samples, for the evaluation of the multiplex PCR.
2.3. Parasite and snail DNA extraction DNA was extracted using the Chelex® DNA extraction technique described by Caron et al. (2011) with modifications. Briefly, a portion (1 mm3 ) of snails and parasites tissue was mixed with 2.5 L of proteinase K (50 mg/mL) (Promega, USA) and 100 L of 5% Chelex® (BioRad USA) using a vortex, and incubated overnight at 56 ◦ C in a T100TM Thermal Cycler (BioRad). Afterwards, the mixture was incubated for 10 min at 95 ◦ C and centrifuged at 10, 000 × g for 5 min. The supernatant was collected and stored at 4 ◦ C, until used. The concentration of DNA from F. hepatica and negative snail samples used during the standardization of the multiplex PCR was measured with a Bio Photometer plus (Eppendorf, Germany) at 260 nm. 2.4. Design of multiplex PCR for F. hepatica–infected G. cubensis detection For the specific amplification of a 340 bp fragment of the ITS-2 of F. hepatica, a set of primers previously described in Ai et al. (2010) (DSFf 5 -ATATTGCGGCCATGGGTTAG-3 and DSJ3 5 CCAATGACAAAGTGACAGCG-3 ) was used. For the co-amplification of G. cubensis 18S rDNA (451 bp), a novel set of primers (GcubF (sense) 5 -GGG GAA GTA TGG TTG CAA AGC-3 and GcubR (antisense) 5 -CCC CAA TCC CTA GCA CGA AG-3 ) was designed based on known sequences of the target gen reported for G. cubensis (Genbank accesion numbers: Z83831, JN614326, JN614327, JN614328, JN614329, JN614330, JN614331, JN614334). The selection of the 18S rDNA primers from these sequences was conducted as follow. Briefly, multiple sequence alignment of the target gen was carried out with the computer program MEGA v5.0 (Tamura et al., 2011). Primers were designed with Primer3 (Rozen and Skaletsky, 2000) to amplify a segment within the 18S rDNA-conserved regions among the Lymnaeidae taxon (Bargues and Mas-Coma, 1997) and to have a melting temperature similar to that of F. hepatica primers. Primers features were calculated by the Oligonucleotide Properties Calculator (Kibbe, 2007) and their primer sequences were compared with available databases to check the specificity with 18S rDNA of other lymnaied snails and to verify possible cross reactions with other species using the BLASTn genomic database (Zhang et al., 2000). 2.5. Optimization of the multiplex PCR In pilot studies using PCR assays, different concentration of F. hepatica primers (0.4 M, 0.8 M and 1.2 M) were evaluated for optimization. Further, different concentrations of G. cubensis primers (0.2 M, 0.4 M, 0.6 M and 0.8 M) were tested in a multiplex PCR in combination with F. hepatica primers at the already optimized concentration. All PCRs were performed in a 25 L reaction mixture using a commercial kit (Qiagen, Germany) containing 1X of Q buffer, 1X Coral Load PCR buffer and a total of 2.5 mM of MgCl2 , 200 M of each deoxynucleoside triphosphate (Eurogentec, Belgium), 1 L of DNA, and 1 U of HotStar Taq® Plus polymerase (Qiagen). During the standardization of the multiplex PCR, 100 ng of G. cubensis DNA were spiked with known amounts of F. hepatica DNA (100 ng and 10 ng). Negative controls (PCR mix with H2 O instead of DNA) were always included, along with positive controls of PCR mix with 20 ng of F. hepatica and 100 ng of non- infected G. cubensis DNA as template. Amplifications of the sequences were performed in a T100TM Thermal Cycler (BioRad) with an initial denaturation step at 95 ◦ C for 5 min, followed by 35 cycles of denaturation at 94 ◦ C for 40 s, annealing at 60 ◦ C for 60 s, polymerization at 72 ◦ C for 60 s, and a final elongation step at 72 ◦ C for 10 min. The amplification products (15 L) were visualized in 2% agarose gels stained with ethidium bromide. The visualization of a band of
Please cite this article in press as: Alba, A., et al., A multiplex PCR for the detection of Fasciola hepatica in the intermediate snail host Galba cubensis. Vet. Parasitol. (2015), http://dx.doi.org/10.1016/j.vetpar.2015.05.012
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approximately 450 bp corresponding to the G. cubensis DNA indicated the absence of PCR inhibitors in the sample. 2.6. Limit of detection and cross reactions The detection limit of the multiplex PCR was assessed on spiked samples consisting of different amounts of G. cubensis DNA (1000 ng, 500 ng, 200 ng, 100 ng, 10 ng), each of them mixed with 1 ng and 0.1 ng of F. hepatica DNA. In addition, 100 ng and 10 ng of G. cubensis DNA were also spiked with 10 ng and 0.01 ng of F. hepatica DNA, respectively. The assay was repeated on two different days. DNA of five trematodes that use lymnaeid snails as intermediate hosts were tested in the multiplex PCR, to assess possible cross reactions of the assay: Fasciola gigantica, Trichobilharzia sp., Cotylurus flabelliformis, Cotylophoron sp. and Echinoparyphium aconiatum. Other known lymnaeid host species of fasciolosis, Pseudosuccinea columella, Radix natalensis, and the planorbid Bulinus truncatus (reported infected with F. hepatica by Hamed et al. (2009)), were also included in the study in order to assess the potential applicability of the developed multiplex PCR for the detection of F. hepatica infection in snail hosts. All DNA were extracted using the same methodology described in Section 2.3 and amplified with the optimized multiplex PCR to assess specificity. 2.7. Assessment of the multiplex PCR with samples from controlled G. cubensis – F. hepatica system The DNA from 66 laboratory-reared G. cubensis as negative samples and from 20 F. hepatica experimentally infectedlaboratory-reared G. cubensis as positive samples was extracted as described in Section 2.3. The parasitological examination of dissected snails was used as reference method for confirming the infection of F. hepatica-exposed G. cubensis. The samples were evaluated with the optimized multiplex PCR using undiluted DNA as a template (Section 2.5). Standard diagnostic indices of the multiplex PCR including sensitivity, specificity and accuracy were calculated at a 95% confidence interval, using the EPIDAT v. 3.1 (2006). 3. Results 3.1. Optimization of the multiplex PCR The optimization of the multiplex PCR mainly involved the standardization of the concentration of both sets of primers. Fig. 1A shows the analytical sensitivity of the F. hepatica-specific PCR using different concentrations of primers. The highest intensity of the electrophoretic band and lower detection limit (0.1 ng) of F. hepatica DNA was assessed with 1.2 M of primers. Therefore, the multiplex PCR was evaluated with spiked samples using 1.2 M of the F. hepatica primer set and different concentrations of G. cubensis primers (Fig. 1B). The intensity of the amplicon corresponding to F. hepatica DNA decreased at increasing concentration of G. cubensis primers (Fig. 1B). The highest resolution of both parasite and snail DNA amplification was achieved with 0.2 M of G. cubensis primers (Fig. 1B) and therefore, this concentration was established for the multiplex PCR together with 1.2 M of F. hepatica primers. 3.2. Limit of detection and cross reactions Once the assay was optimized, the detection limit of parasite DNA was assessed in samples spiked with different amounts of G. cubensis DNA. The multiplex PCR was able to detect 0.1 ng of F. hepatica DNA even at high concentrations of snail DNA (1000 ng) (see Fig. 2). In addition, when parasite DNA was spiked on low amounts of G. cubensis DNA (e.g., 10 ng) amplification of 0.01 ng of F. hepatica
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DNA was recorded. Similar results were achieved when the assay was repeated on two different days. Fig. 3 shows the results of the evaluation of the multiplex PCR with other snails reported as intermediate hosts of Fasciola spp. and with trematode species that use lymnaeid snails as intermediate hosts. No amplification of the planorbid B. truncatus and the fasciolid F. gigantica DNA was observed. However, a band between 400 and 500 bp was displayed with the other tested species in the multiplex PCR (Fig. 3). Amplicons of similar sizes were expected with P. columella and R. natalensis since 18S primers were selected within a conserved region of this gen. However, due to its size (similar to the size of G. cubensis amplicon) the amplified band with the related snails and trematodes has no impact on the interpretation of the multiplex PCR. 3.3. Assessment of the multiplex PCR on samples of experimental G. cubensis – F. hepatica system The multiplex PCR was evaluated with laboratory-reared snails in order to assess the sensitivity and specificity of the assay. All non-infected G. cubensis (66) were tested by the PCR and were negative for parasite infection while all 20 confirmed F. hepaticainfected snails displayed two bands of expected size both the snail and parasite DNA (Fig. 4). No inhibition of the reaction occurred since the amplicon of expected size around 450 bp (corresponding to the size of the target fragment of G. cubensis 18S rDNA used as internal control) was always recorded. Standard diagnostic indices were calculated to assess the multiplex PCR and resulted in: 100% (97.5–100%) of sensitivity, 100% (99.24–100%) of specificity and 100% (99.42–100%) of accuracy. 4. Discussion F. hepatica require particular mollusc intermediate host for the development of larval stages and this dependence can be used to control the transmission of the disease. The knowledge of the presence of the parasite in its snail hosts of a particular area is a key element to detect possible transmission foci and to estimate the infection risk. However, the prevalence of F. hepatica in naturally infected intermediate host snails is usually low. Therefore, it is essential to apply reliable methods to survey snail populations in risk areas. Microscopic examination of parasite larvae after snail dissection and/or cercarial shedding is still widely used (Dreyfuss et al., 2005; Kleiman et al., 2007). Nevertheless, there are two drawbacks of major importance when dealing with techniques based on microscopy: (1) detection of early infection is difficult (due to the small size of the larva and its localization within the snail) while monitoring of cercarial shedding is time-consuming and would require laboratory rearing of snails, and (2) uncertainties regarding the identification of parasite species based only on intramolluscan larval morphology may arise (Caron et al., 2008). A number of molecular assays have been developed to detect F. hepatica in different intermediate hosts species (Kaplan et al., 1997; Cucher et al., 2006; Caron et al., 2011) using several target sequences. Kaplan et al. (1995) reported the identification of a specific 124 bp fragment of repetitive Fasciola spp. DNA that constitutes approximately 15% of the genome of the parasite. This fragment was further used in the development of a DNA probe assay to detect F. hepatica in field-collected G. cubensis (Kaplan et al., 1997). With the development of PCR-based methods, this target has been widely used in PCR-based assays devised for amplification of F. hepatica and F. gigantica within different intermediate host species (i.e., ˛ Velusamy et al., 2004; Kozac and Wedrychowicz, 2010; Caron et al., 2011). Magalhães et al. (2004,2008); Magalhães et al. (2004,2008) used an 85 bp-repetitive tandem sequence of F. hepatica mitochon-
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Fig. 1. (A) Agarose gel showing the amplicons of 340 bp produced in a PCR with concentrations of Fasciola hepatica primers of 0.4 M, 0.8 M and 1.2 M. Lane headings represents the amounts of F. hepatica DNA used as template (values in ng); C− : DNA-free reaction mixture used as negative control. MM: Molecular weight marker GeneRulerTM 100 bp DNA ladder (MBI Fermentas). (B) Agarose gel showing the amplicons produced with the multiplex PCR using 1.2 M of F. hepatica primers and 0.2 M, 0.4 M, 0.6 M and 0.8 M of Galba cubensis primers. Lane headings represents the amounts of F. hepatica DNA/amounts of G. cubensis DNA used as template (values in ng) at different concentrations of G. cubensis primers; F.hep+ : Positive control of F. hepatica template DNA where the expected amplicon of 340 bp is observed; G.cub+ : G. cubensis template DNA where the expected amplicon of 451 bp is observed; C− : DNA-free reaction mixture used as negative control. MM: Molecular weight marker GeneRulerTM 100 bp DNA ladder.
Fig. 2. Agarose gel following multiplex PCR to illustrate the detection limit of Fasciola hepatica on spiked samples of Galba cubensis. Lane headings represents the amounts of F. hepatica DNA/amounts of G. cubensis DNA used as template (values in ng); G.cub+ : G. cubensis template DNA where the expected amplicon of 451 bp is observed; F.hep+ : Positive control of F. hepatica template DNA where the expected amplicon of 340 bp is observed; C− : DNA-free reaction mixture used as negative control. MM: Molecular weight marker GeneRulerTM 100 bp DNA ladder.
Fig. 3. Agarose gel following multiplex PCR to illustrate possible cross reactions with related trematodes and snails. Lane 1: Pseudosuccinea columella; Lane 2: Radix natalensis; Lane 3: Bulinus truncatus; Lane 4: Fasciola gigantica; Lane 5: Trichobilharzia sp.; Lane 6: Cotylurus flabelliformis; Lane 7: Cotylophoron sp.; Lane 8: Echinoparyphium aconiatum; G.cub+ : Galba cubensis template DNA where the expected amplicon of 451 bp is observed; F.hep+ : Positive control of Fasciola hepatica template DNA where the expected amplicon of 340 bp is observed; C− : DNA-free reaction mixture used as negative control. MM: Molecular weight marker GeneRulerTM 100 bp DNA ladder.
Fig. 4. Agarose gel showing the amplicons produced in the multiplex PCR with some of the negative and Fasciola hepatica-positive Galba cubensis samples. 1–3: non-infected laboratory-reared G. cubensis; Lane 4–10: F. hepatica-infected laboratory-reared G. cubensis (infection confirmed by parasitological examination); lane; Gcub+ : G. cubensis template DNA where the expected amplicon of 451 bp is observed. F.hep+ : Positive control of F. hepatica template DNA where the expected amplicon of 340 bp is observed; C− : DNA-free reaction mixture used as negative control. MM: Molecular weight marker GeneRulerTM 100 bp DNA ladder.
drial DNA for parasite detection in a multiplex PCR which, as in the case of the amplification of the 124 bp-repetitive segments of Fasciola spp., displays a ladder pattern of multiple DNA fragments. Furthermore, segments of the multi-copy genes of the mitochondrial cytochrome c oxidase subunit I (Cucher et al., 2006) and the nuclear ITS-2 rDNA of F. hepatica (Ai et al., 2010) have also been used as target for detecting parasite-infected snails through PCRs. Here, we propose the first multiplex PCR method for detecting F. hepatica in the snail G. cubensis. The selected technique allows the amplification of several sequences in the same PCR tube and therefore, the amplification of an internal control (i.e., 18S rDNA of G. cubensis) along with the gene of interest (ITS-2 of F. hepatica)
reduce the possibility of false negative results due to the presence of PCR inhibitors in a given sample. In fact, the presence of remnants on snail samples that interfere with DNA amplification has been reported and was overcome by adding bovine serum albumin (Cucher et al., 2006). The optimization of the multiplex PCR showed interesting results since the concentration of the primers turned out to be a key factor. Ai et al. (2010) reported a PCR for the identification and discrimination of Fasciola species using 2 M of F. hepaticaspecific primers. The smallest amount of F. hepatica DNA that was detectable by these authors was 0.11 ng. In our experimental conditions we reached a sensitivity of 0.1 ng of F. hepatica DNA using a
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lower concentration of primers (1.2 M). In addition, the observation of both bands (snail and parasite DNA) in the multiplex PCR was obtained at 0.2 M of G. cubensis primers, while at higher concentrations a preferential amplification of snail DNA always occurred. Differences regarding the concentration ratio between both set of primers in a multiplex PCR can also be observed in the assay developed by Caron et al. (2011) for the detection of Fasciola spp. in G. truncatula. In contrast to our results, these authors used snailspecific primers highly concentrated (50 M) and only 5 M of Fasciola spp. primers. The limit for detecting F. hepatica DNA in the developed PCR (0.1 ng) is five to ten fold higher than the DNA concentration of a single miracidium (0.5–1 ng) (Kaplan et al., 1997; Magalhães et al., 2004). This analytical sensitivity remained even at high concentrations of G. cubensis DNA, and enables the detection of the parasite during all periods of the infection in snails, since one miracidium constitutes the minimal biological infestation unit of the intermediate host. The amplification of the 18S rDNA of P. columella and R. natalensis was predicted from the in silico analysis since the target segment used in the assay for G. cubensis detection is conserved among different populations of these species and among several lymnaeid snails (e.g., G. truncatula, Galba viatrix and Omphiscola glabra). Taking into account that the devised multiplex PCR is aimed at detecting F. hepatica within the intermediate host we judged it convenient to use a conserved sequence among the Lymnaeidae taxon as internal control of the reaction. Therefore, the developed assay could be adapted to other lymnaeid hosts of fasciolosis as a unique tool for the surveillance of F. hepatica transmission. Amplification of the 18S rDNA of other digenean trematodes was predicted as well from the in silico analysis, with amplicon sizes between 400 and 600 bp. The recorded DNA amplification with related snails and trematodes has no impact on the interpretation of the multiplex PCR (i.e., detection of F. hepatica in snails) because DNA fragments produced with these species are of a different size that the amplified segment of F. hepatica ITS-2. The multiplex PCR proved useful in the detection of F. hepatica infection in G. cubensis and showed total agreement with the reference method in discriminating positive and negative samples. Further studies on field populations of G. cubensis are needed to assess the suitability of the method. However, the developed assay constitutes a new and reliable tool for the survey of parasite transmission through one of its main intermediate hosts in the American region. 5. Conclusion The first multiplex PCR assay for the detection of F. hepatica in the intermediate host G. cubensis was developed. The method showed a limit of detection of parasite DNA equivalent to less than one miracidium, and it could be applied to other F. hepatica-infected lymnaeid snail species. Conflict of interest The authors declare that there are no conflicts of interest. Acknowledgments Financial support for this investigation was provided by a 2014–2015 grant from the Strategic Network on Neglected Diseases and Zoonoses from the Institute of Tropical Medicine (ITM, Antwerp, Belgium), funded by the framework agreement with the Belgian Directorate General for Development. Authors would like to thank fellow colleagues from Angola for providing us with the
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A multiplex PCR for the detection of Fasciola hepatica in the intermediate snail host Galba cubensis Annia Alba a , Antonio A. Vázquez b , Hilda Hernández a , Jorge Sánchez b , Ricardo Marcet a , Mabel Figueredo a , Jorge Sarracent a , Jorge Fraga c,∗ a Laboratorio de Anticuerpos Monoclonales, Departamento de Parasitología, Instituto de Medicina Tropical “Pedro Kourí”, Ave Novia del Mediodía km 6 ½, AP 601, La Habana, Cuba b Laboratorio de Malacología, Departamento de Control de Vectores, Instituto de Medicina Tropical “Pedro Kourí”, Ave Novia del Mediodía km 6 ½, AP 601, La Habana, Cuba c Laboratorio de Biología Molecular, Departamento de Parasitología, Instituto de Medicina Tropical “Pedro Kourí”, Ave Novia del Mediodía km 6 ½, AP 601, La Habana, Cuba
a r t i c l e
i n f o
Article history: Received 23 October 2014 Received in revised form 1 May 2015 Accepted 9 May 2015 Keywords: Fasciola hepatica Galba cubensis Multiplex PCR Epidemiology Diagnosis
a b s t r a c t Fasciolosis is a snail-borne trematode infection that has re-emerged as a human disease, and is considered a significant problem for veterinary medicine worldwide. The evaluation of the transmission risk of fasciolosis as well as the efficacy of the strategies for its control could be carried out through epidemiological surveillance of the snails that act as intermediate hosts of the parasites. The present study aimed to develop the first multiplex PCR to detect Fasciola hepatica in Galba cubensis, an important intermediate host of the parasite in the Americas and especially in the Caribbean basin. The multiplex PCR was optimized for the amplification of a 340 bp fragment of the second internal transcribed spacer (ITS-2) of F. hepatica rDNA, while another set of primers was designed and used to amplify a conserved segment of the nuclear 18S rDNA of the snail (451 bp), as an internal control of the reaction. The assay was able to detect up to 100 pg of the parasite even at high concentrations of snail DNA, an analytical sensitivity that allows the detection of less than a single miracidium, which is the minimal biological infestation unit. A controlled laboratory-reared G. cubensis – F. hepatica system was used for the evaluation of the developed multiplex PCR, and 100% sensitivity and specificity was achieved. This assay constitutes a novel, useful and suitable technique for the survey of fasciolosis transmission through one of the main intermediate hosts in the Western hemisphere. © 2015 Elsevier B.V. All rights reserved.
1. Introduction The liver fluke Fasciola hepatica is a globally distributed trematode and the main causative agent of fasciolosis (Mas-Coma et al., 2009). The parasite is characterized by a complex life cycle that includes switching through several larval stages and the development inside two very different hosts. Adults of F. hepatica develop inside mammals where they can live for years feeding on the host and laying thousands of eggs per day (Andrews, 1999). A variety of domestic mammals (e.g., sheep, cattle, buffaloes) can act as definitive hosts of F. hepatica and in fact, high prevalence of the parasite in these animals is reported worldwide (Mas-Coma et al., 2009). Consequently, fasciolosis is a serious animal health problem that
∗ Corresponding author. Tel.: +53 7 255 3112; fax: +53 7 204 6051. E-mail address: [email protected] (J. Fraga).
causes significant economic losses in several developed and developing countries (Kaplan, 2001; Espinoza et al., 2010). Humans can be affected by this parasite as well, acting as definitive hosts, and in a number of countries the disease is a significant problem for public health (Mas-Coma et al., 2009). The development of several larval stages of the parasite occurs inside freshwater snails of the family Lymnaeidae, which act as intermediate hosts. Hence, if snails occurring in the field (e.g., grazing pastures and flooded crops such as watercress and rice fields) are discovered infected with F. hepatica, direct control strategies aimed to reduce their populations or avoid contact with definitive hosts will mostly control transmission. The surveillance of high risks areas where intermediate and definitive hosts occur could help to prevent or control potential outbreaks. There are many lymnaeid species known as intermediate hosts of fasciolosis worldwide (Correa et al., 2011). The transmission of the disease is dependent on the lymnaeid hosts that occur in a par-
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ticular geographical area and their roles regarding transmission is influenced by the local adaptation of the species (Dar et al., 2013; Vázquez et al., 2014). In the Caribbean area, the lymnaeid snail Galba cubensis (formerly known as Lymnaea (Fossaria) cubensis) is considered the main intermediate host of F. hepatica (Gutiérrez et al., 2011). However, its presence in some regions of North (Kaplan et al., 1997) and South America (Bargues et al., 2011; Medeiros et al., 2014), where it co-exist with other snail hosts complicates the epidemiological scenario of fasciolosis. Several investigations have been conducted, mainly in Cuba where fasciolosis is endemic in livestock, regarding the role of G. cubensis in F. hepatica trans˜ mission (Vázquez et al., 2014), its ecology and distribution (Canete et al., 2004; Vázquez et al., 2009), and control strategies for its management (Perera et al., 1991). A DNA probe assay was developed and validated in field-collected G. cubensis (Kaplan et al., 1997) but the complicated and time-consuming technique of DNA hybridization could limit its application during large-scale surveys of F. hepatica infection in snails. Moreover, several PCR-based methods have been developed to detect F. hepatica infection in snail hosts (i.e., Magalhães et al., 2004; Cucher et al., 2006; Caron et al., 2011) but to our knowledge there are no studies reporting these PCR techniques on G. cubensis. So far, the detection of the parasite in this particular intermediate host mainly relies on the classical parasitological examination of field-collected snails. The aim of this study was to develop the first multiplex PCR to detect F. hepatica-infected G. cubensis. A previously reported set of primers was used to amplify a fragment of the second internal transcribed spacer (ITS-2) within the F. hepatica ribosomal DNA (rDNA). In addition, a novel set of primers was designed to amplify a conserved segment of the 18S rDNA of G. cubensis and other snails from the Lymnaeidae taxon, in order to use it in the assay as an internal control. The method was evaluated on samples of experimental snail-parasite system using laboratory-reared G. cubensis populations.
2. Material and methods 2.1. Parasites and snails Adults of F. hepatica were collected from the livers of infected cattle and placed in saline solution (0.85% NaCl) supplemented with 5% glucose for transport and egg laying. Identification of recovered flukes was done following Jones (2005). Eggs were then thoroughly washed and preserved in saline solution with Gentamicin, at 4 ◦ C until use. Adults of F. hepatica were preserved in 95% ethanol. G. cubensis was reared in the Laboratory of Malacology of the Institute of Tropical Medicine “Pedro Kourí” according to the methodology of Sánchez et al. (1995). Subsequent generations, considered free of infection, were used as negative controls. Some of the laboratory-produced snails were also used in experimental infection with F. hepatica miracidia to obtain positive control samples.
2.2. Experimental infection of G. cubensis F. hepatica eggs (see Section 2.1) were placed in distilled water and were incubated at 28 ◦ C, for 15 days in the dark. Miracidia were obtained after egg hatching induced by light exposure and experimental infection of 50 laboratory-reared G. cubensis was conducted as described by Vázquez et al. (2014). Snails were analyzed for intramolluscan larvae of F. hepatica by parasitological examination of dissected individuals (Caron et al., 2008) starting from day 14 post-exposure. Only the infected snails were preserved in 95% ethanol as positive samples, for the evaluation of the multiplex PCR.
2.3. Parasite and snail DNA extraction DNA was extracted using the Chelex® DNA extraction technique described by Caron et al. (2011) with modifications. Briefly, a portion (1 mm3 ) of snails and parasites tissue was mixed with 2.5 L of proteinase K (50 mg/mL) (Promega, USA) and 100 L of 5% Chelex® (BioRad USA) using a vortex, and incubated overnight at 56 ◦ C in a T100TM Thermal Cycler (BioRad). Afterwards, the mixture was incubated for 10 min at 95 ◦ C and centrifuged at 10, 000 × g for 5 min. The supernatant was collected and stored at 4 ◦ C, until used. The concentration of DNA from F. hepatica and negative snail samples used during the standardization of the multiplex PCR was measured with a Bio Photometer plus (Eppendorf, Germany) at 260 nm. 2.4. Design of multiplex PCR for F. hepatica–infected G. cubensis detection For the specific amplification of a 340 bp fragment of the ITS-2 of F. hepatica, a set of primers previously described in Ai et al. (2010) (DSFf 5 -ATATTGCGGCCATGGGTTAG-3 and DSJ3 5 CCAATGACAAAGTGACAGCG-3 ) was used. For the co-amplification of G. cubensis 18S rDNA (451 bp), a novel set of primers (GcubF (sense) 5 -GGG GAA GTA TGG TTG CAA AGC-3 and GcubR (antisense) 5 -CCC CAA TCC CTA GCA CGA AG-3 ) was designed based on known sequences of the target gen reported for G. cubensis (Genbank accesion numbers: Z83831, JN614326, JN614327, JN614328, JN614329, JN614330, JN614331, JN614334). The selection of the 18S rDNA primers from these sequences was conducted as follow. Briefly, multiple sequence alignment of the target gen was carried out with the computer program MEGA v5.0 (Tamura et al., 2011). Primers were designed with Primer3 (Rozen and Skaletsky, 2000) to amplify a segment within the 18S rDNA-conserved regions among the Lymnaeidae taxon (Bargues and Mas-Coma, 1997) and to have a melting temperature similar to that of F. hepatica primers. Primers features were calculated by the Oligonucleotide Properties Calculator (Kibbe, 2007) and their primer sequences were compared with available databases to check the specificity with 18S rDNA of other lymnaied snails and to verify possible cross reactions with other species using the BLASTn genomic database (Zhang et al., 2000). 2.5. Optimization of the multiplex PCR In pilot studies using PCR assays, different concentration of F. hepatica primers (0.4 M, 0.8 M and 1.2 M) were evaluated for optimization. Further, different concentrations of G. cubensis primers (0.2 M, 0.4 M, 0.6 M and 0.8 M) were tested in a multiplex PCR in combination with F. hepatica primers at the already optimized concentration. All PCRs were performed in a 25 L reaction mixture using a commercial kit (Qiagen, Germany) containing 1X of Q buffer, 1X Coral Load PCR buffer and a total of 2.5 mM of MgCl2 , 200 M of each deoxynucleoside triphosphate (Eurogentec, Belgium), 1 L of DNA, and 1 U of HotStar Taq® Plus polymerase (Qiagen). During the standardization of the multiplex PCR, 100 ng of G. cubensis DNA were spiked with known amounts of F. hepatica DNA (100 ng and 10 ng). Negative controls (PCR mix with H2 O instead of DNA) were always included, along with positive controls of PCR mix with 20 ng of F. hepatica and 100 ng of non- infected G. cubensis DNA as template. Amplifications of the sequences were performed in a T100TM Thermal Cycler (BioRad) with an initial denaturation step at 95 ◦ C for 5 min, followed by 35 cycles of denaturation at 94 ◦ C for 40 s, annealing at 60 ◦ C for 60 s, polymerization at 72 ◦ C for 60 s, and a final elongation step at 72 ◦ C for 10 min. The amplification products (15 L) were visualized in 2% agarose gels stained with ethidium bromide. The visualization of a band of
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approximately 450 bp corresponding to the G. cubensis DNA indicated the absence of PCR inhibitors in the sample. 2.6. Limit of detection and cross reactions The detection limit of the multiplex PCR was assessed on spiked samples consisting of different amounts of G. cubensis DNA (1000 ng, 500 ng, 200 ng, 100 ng, 10 ng), each of them mixed with 1 ng and 0.1 ng of F. hepatica DNA. In addition, 100 ng and 10 ng of G. cubensis DNA were also spiked with 10 ng and 0.01 ng of F. hepatica DNA, respectively. The assay was repeated on two different days. DNA of five trematodes that use lymnaeid snails as intermediate hosts were tested in the multiplex PCR, to assess possible cross reactions of the assay: Fasciola gigantica, Trichobilharzia sp., Cotylurus flabelliformis, Cotylophoron sp. and Echinoparyphium aconiatum. Other known lymnaeid host species of fasciolosis, Pseudosuccinea columella, Radix natalensis, and the planorbid Bulinus truncatus (reported infected with F. hepatica by Hamed et al. (2009)), were also included in the study in order to assess the potential applicability of the developed multiplex PCR for the detection of F. hepatica infection in snail hosts. All DNA were extracted using the same methodology described in Section 2.3 and amplified with the optimized multiplex PCR to assess specificity. 2.7. Assessment of the multiplex PCR with samples from controlled G. cubensis – F. hepatica system The DNA from 66 laboratory-reared G. cubensis as negative samples and from 20 F. hepatica experimentally infectedlaboratory-reared G. cubensis as positive samples was extracted as described in Section 2.3. The parasitological examination of dissected snails was used as reference method for confirming the infection of F. hepatica-exposed G. cubensis. The samples were evaluated with the optimized multiplex PCR using undiluted DNA as a template (Section 2.5). Standard diagnostic indices of the multiplex PCR including sensitivity, specificity and accuracy were calculated at a 95% confidence interval, using the EPIDAT v. 3.1 (2006). 3. Results 3.1. Optimization of the multiplex PCR The optimization of the multiplex PCR mainly involved the standardization of the concentration of both sets of primers. Fig. 1A shows the analytical sensitivity of the F. hepatica-specific PCR using different concentrations of primers. The highest intensity of the electrophoretic band and lower detection limit (0.1 ng) of F. hepatica DNA was assessed with 1.2 M of primers. Therefore, the multiplex PCR was evaluated with spiked samples using 1.2 M of the F. hepatica primer set and different concentrations of G. cubensis primers (Fig. 1B). The intensity of the amplicon corresponding to F. hepatica DNA decreased at increasing concentration of G. cubensis primers (Fig. 1B). The highest resolution of both parasite and snail DNA amplification was achieved with 0.2 M of G. cubensis primers (Fig. 1B) and therefore, this concentration was established for the multiplex PCR together with 1.2 M of F. hepatica primers. 3.2. Limit of detection and cross reactions Once the assay was optimized, the detection limit of parasite DNA was assessed in samples spiked with different amounts of G. cubensis DNA. The multiplex PCR was able to detect 0.1 ng of F. hepatica DNA even at high concentrations of snail DNA (1000 ng) (see Fig. 2). In addition, when parasite DNA was spiked on low amounts of G. cubensis DNA (e.g., 10 ng) amplification of 0.01 ng of F. hepatica
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DNA was recorded. Similar results were achieved when the assay was repeated on two different days. Fig. 3 shows the results of the evaluation of the multiplex PCR with other snails reported as intermediate hosts of Fasciola spp. and with trematode species that use lymnaeid snails as intermediate hosts. No amplification of the planorbid B. truncatus and the fasciolid F. gigantica DNA was observed. However, a band between 400 and 500 bp was displayed with the other tested species in the multiplex PCR (Fig. 3). Amplicons of similar sizes were expected with P. columella and R. natalensis since 18S primers were selected within a conserved region of this gen. However, due to its size (similar to the size of G. cubensis amplicon) the amplified band with the related snails and trematodes has no impact on the interpretation of the multiplex PCR. 3.3. Assessment of the multiplex PCR on samples of experimental G. cubensis – F. hepatica system The multiplex PCR was evaluated with laboratory-reared snails in order to assess the sensitivity and specificity of the assay. All non-infected G. cubensis (66) were tested by the PCR and were negative for parasite infection while all 20 confirmed F. hepaticainfected snails displayed two bands of expected size both the snail and parasite DNA (Fig. 4). No inhibition of the reaction occurred since the amplicon of expected size around 450 bp (corresponding to the size of the target fragment of G. cubensis 18S rDNA used as internal control) was always recorded. Standard diagnostic indices were calculated to assess the multiplex PCR and resulted in: 100% (97.5–100%) of sensitivity, 100% (99.24–100%) of specificity and 100% (99.42–100%) of accuracy. 4. Discussion F. hepatica require particular mollusc intermediate host for the development of larval stages and this dependence can be used to control the transmission of the disease. The knowledge of the presence of the parasite in its snail hosts of a particular area is a key element to detect possible transmission foci and to estimate the infection risk. However, the prevalence of F. hepatica in naturally infected intermediate host snails is usually low. Therefore, it is essential to apply reliable methods to survey snail populations in risk areas. Microscopic examination of parasite larvae after snail dissection and/or cercarial shedding is still widely used (Dreyfuss et al., 2005; Kleiman et al., 2007). Nevertheless, there are two drawbacks of major importance when dealing with techniques based on microscopy: (1) detection of early infection is difficult (due to the small size of the larva and its localization within the snail) while monitoring of cercarial shedding is time-consuming and would require laboratory rearing of snails, and (2) uncertainties regarding the identification of parasite species based only on intramolluscan larval morphology may arise (Caron et al., 2008). A number of molecular assays have been developed to detect F. hepatica in different intermediate hosts species (Kaplan et al., 1997; Cucher et al., 2006; Caron et al., 2011) using several target sequences. Kaplan et al. (1995) reported the identification of a specific 124 bp fragment of repetitive Fasciola spp. DNA that constitutes approximately 15% of the genome of the parasite. This fragment was further used in the development of a DNA probe assay to detect F. hepatica in field-collected G. cubensis (Kaplan et al., 1997). With the development of PCR-based methods, this target has been widely used in PCR-based assays devised for amplification of F. hepatica and F. gigantica within different intermediate host species (i.e., ˛ Velusamy et al., 2004; Kozac and Wedrychowicz, 2010; Caron et al., 2011). Magalhães et al. (2004,2008); Magalhães et al. (2004,2008) used an 85 bp-repetitive tandem sequence of F. hepatica mitochon-
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Fig. 1. (A) Agarose gel showing the amplicons of 340 bp produced in a PCR with concentrations of Fasciola hepatica primers of 0.4 M, 0.8 M and 1.2 M. Lane headings represents the amounts of F. hepatica DNA used as template (values in ng); C− : DNA-free reaction mixture used as negative control. MM: Molecular weight marker GeneRulerTM 100 bp DNA ladder (MBI Fermentas). (B) Agarose gel showing the amplicons produced with the multiplex PCR using 1.2 M of F. hepatica primers and 0.2 M, 0.4 M, 0.6 M and 0.8 M of Galba cubensis primers. Lane headings represents the amounts of F. hepatica DNA/amounts of G. cubensis DNA used as template (values in ng) at different concentrations of G. cubensis primers; F.hep+ : Positive control of F. hepatica template DNA where the expected amplicon of 340 bp is observed; G.cub+ : G. cubensis template DNA where the expected amplicon of 451 bp is observed; C− : DNA-free reaction mixture used as negative control. MM: Molecular weight marker GeneRulerTM 100 bp DNA ladder.
Fig. 2. Agarose gel following multiplex PCR to illustrate the detection limit of Fasciola hepatica on spiked samples of Galba cubensis. Lane headings represents the amounts of F. hepatica DNA/amounts of G. cubensis DNA used as template (values in ng); G.cub+ : G. cubensis template DNA where the expected amplicon of 451 bp is observed; F.hep+ : Positive control of F. hepatica template DNA where the expected amplicon of 340 bp is observed; C− : DNA-free reaction mixture used as negative control. MM: Molecular weight marker GeneRulerTM 100 bp DNA ladder.
Fig. 3. Agarose gel following multiplex PCR to illustrate possible cross reactions with related trematodes and snails. Lane 1: Pseudosuccinea columella; Lane 2: Radix natalensis; Lane 3: Bulinus truncatus; Lane 4: Fasciola gigantica; Lane 5: Trichobilharzia sp.; Lane 6: Cotylurus flabelliformis; Lane 7: Cotylophoron sp.; Lane 8: Echinoparyphium aconiatum; G.cub+ : Galba cubensis template DNA where the expected amplicon of 451 bp is observed; F.hep+ : Positive control of Fasciola hepatica template DNA where the expected amplicon of 340 bp is observed; C− : DNA-free reaction mixture used as negative control. MM: Molecular weight marker GeneRulerTM 100 bp DNA ladder.
Fig. 4. Agarose gel showing the amplicons produced in the multiplex PCR with some of the negative and Fasciola hepatica-positive Galba cubensis samples. 1–3: non-infected laboratory-reared G. cubensis; Lane 4–10: F. hepatica-infected laboratory-reared G. cubensis (infection confirmed by parasitological examination); lane; Gcub+ : G. cubensis template DNA where the expected amplicon of 451 bp is observed. F.hep+ : Positive control of F. hepatica template DNA where the expected amplicon of 340 bp is observed; C− : DNA-free reaction mixture used as negative control. MM: Molecular weight marker GeneRulerTM 100 bp DNA ladder.
drial DNA for parasite detection in a multiplex PCR which, as in the case of the amplification of the 124 bp-repetitive segments of Fasciola spp., displays a ladder pattern of multiple DNA fragments. Furthermore, segments of the multi-copy genes of the mitochondrial cytochrome c oxidase subunit I (Cucher et al., 2006) and the nuclear ITS-2 rDNA of F. hepatica (Ai et al., 2010) have also been used as target for detecting parasite-infected snails through PCRs. Here, we propose the first multiplex PCR method for detecting F. hepatica in the snail G. cubensis. The selected technique allows the amplification of several sequences in the same PCR tube and therefore, the amplification of an internal control (i.e., 18S rDNA of G. cubensis) along with the gene of interest (ITS-2 of F. hepatica)
reduce the possibility of false negative results due to the presence of PCR inhibitors in a given sample. In fact, the presence of remnants on snail samples that interfere with DNA amplification has been reported and was overcome by adding bovine serum albumin (Cucher et al., 2006). The optimization of the multiplex PCR showed interesting results since the concentration of the primers turned out to be a key factor. Ai et al. (2010) reported a PCR for the identification and discrimination of Fasciola species using 2 M of F. hepaticaspecific primers. The smallest amount of F. hepatica DNA that was detectable by these authors was 0.11 ng. In our experimental conditions we reached a sensitivity of 0.1 ng of F. hepatica DNA using a
Please cite this article in press as: Alba, A., et al., A multiplex PCR for the detection of Fasciola hepatica in the intermediate snail host Galba cubensis. Vet. Parasitol. (2015), http://dx.doi.org/10.1016/j.vetpar.2015.05.012
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lower concentration of primers (1.2 M). In addition, the observation of both bands (snail and parasite DNA) in the multiplex PCR was obtained at 0.2 M of G. cubensis primers, while at higher concentrations a preferential amplification of snail DNA always occurred. Differences regarding the concentration ratio between both set of primers in a multiplex PCR can also be observed in the assay developed by Caron et al. (2011) for the detection of Fasciola spp. in G. truncatula. In contrast to our results, these authors used snailspecific primers highly concentrated (50 M) and only 5 M of Fasciola spp. primers. The limit for detecting F. hepatica DNA in the developed PCR (0.1 ng) is five to ten fold higher than the DNA concentration of a single miracidium (0.5–1 ng) (Kaplan et al., 1997; Magalhães et al., 2004). This analytical sensitivity remained even at high concentrations of G. cubensis DNA, and enables the detection of the parasite during all periods of the infection in snails, since one miracidium constitutes the minimal biological infestation unit of the intermediate host. The amplification of the 18S rDNA of P. columella and R. natalensis was predicted from the in silico analysis since the target segment used in the assay for G. cubensis detection is conserved among different populations of these species and among several lymnaeid snails (e.g., G. truncatula, Galba viatrix and Omphiscola glabra). Taking into account that the devised multiplex PCR is aimed at detecting F. hepatica within the intermediate host we judged it convenient to use a conserved sequence among the Lymnaeidae taxon as internal control of the reaction. Therefore, the developed assay could be adapted to other lymnaeid hosts of fasciolosis as a unique tool for the surveillance of F. hepatica transmission. Amplification of the 18S rDNA of other digenean trematodes was predicted as well from the in silico analysis, with amplicon sizes between 400 and 600 bp. The recorded DNA amplification with related snails and trematodes has no impact on the interpretation of the multiplex PCR (i.e., detection of F. hepatica in snails) because DNA fragments produced with these species are of a different size that the amplified segment of F. hepatica ITS-2. The multiplex PCR proved useful in the detection of F. hepatica infection in G. cubensis and showed total agreement with the reference method in discriminating positive and negative samples. Further studies on field populations of G. cubensis are needed to assess the suitability of the method. However, the developed assay constitutes a new and reliable tool for the survey of parasite transmission through one of its main intermediate hosts in the American region. 5. Conclusion The first multiplex PCR assay for the detection of F. hepatica in the intermediate host G. cubensis was developed. The method showed a limit of detection of parasite DNA equivalent to less than one miracidium, and it could be applied to other F. hepatica-infected lymnaeid snail species. Conflict of interest The authors declare that there are no conflicts of interest. Acknowledgments Financial support for this investigation was provided by a 2014–2015 grant from the Strategic Network on Neglected Diseases and Zoonoses from the Institute of Tropical Medicine (ITM, Antwerp, Belgium), funded by the framework agreement with the Belgian Directorate General for Development. Authors would like to thank fellow colleagues from Angola for providing us with the
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