INTRODUCTION

Fasciola hepatica (liver fluke) is a parasitic trematode that causes fascioliasis, a worldwide distributed disease that affects domestic livestock and humans (Mas-Coma et al., 2009). In livestock industry, fascioliasis causes important economic losses as it leads to a reduction in the production of meat, milk, or wool. The direct economic impact of the disease is increased condemnation of liver meat, but the far more damaging effects are a reduction in animal productivity, lower birth weight, and reduced growth of infected animals (Howell et al., 2015; Khoramian et al., 2014). F. hepatica is also an important human pathogen and fascioliasis is considered a re-emerging parasitic disease in many countries (Mas-Coma et al., 2008).

The life cycle of F. hepatica involves lymnaeid snails as intermediate hosts and depends on the development of larval stages. Detection of F. hepatica in lymnaeid snails is a helpful tool to provide information on the level of pasture contamination and for prevalence studies, which are impor­tant issues in the control of fascioliasis in livestock (Caron et al., 2008). Classical laboratory diagnosis for the identification of larval stages of F. hepatica in lymnaeid snails involves snail crushing and examination under an optical microscope (Caron et al., 2008). This method is widely used because of its simplicity and the low cost of materials and equipment required. However, for a highly specific result, the method must be carried out by an experienced technician and its sensitivity is relatively low (Caron et al., 2008).

To improve the identification and characterization of F. hepatica in snails, molecular techniques are increasingly being developed (Alba et al., 2015; Caron et al., 2011; Cucher et al., 2006; Kozak and We, 2010; Magalhães et al., 2004; Velusamy et al., 2004). Although molecular techniques require specialized equipment, they may help overcome the specificity and sensitivity problems. However, the presence of false-positive and false-negative results is a key issue to be considered in the development of any reliable molecular technique (Burkardt, 2000; Victor et al., 1993). Indeterminated results may be due to the contamination of negative samples or the presence of PCR inhibitors.

The aim of this work was to develop a multiplex PCR to detect F. hepatica in snails to improve the sensitivity and the spe­cificity, to minimize false-negative and false-positive results.

MATERIALS AND METHODS

Field-collected snails and parasite materials

Adult F. hepatica flukes were obtained from the liver of a naturally infected sheep. Eggs of F. hepatica were recove­red from fecal samples of naturally infected animals and incuba­ted in the dark at 26ºC for 14 days to isolate miracidia. Embryonated eggs were observed under a microscope until the release of miracidia. Each miracidium was preserved indivi­dually in tubes with 70% alcohol at -20°C until DNA extraction.

Snails were collected from bodies of water in the province of Neuquén, Argentina, during summertime. The snails were examined under the miscrocope to determine the presence of trematode larvae, as previously reported, and conserved in 70% ethanol at -20ºC until use (Prepelitchi et al., 2003).

To compare the duplex PCR and microscopic exami­nation, these techniques were conducted in parallel in field-collected snails. Also, we compared both techniques in two sets of snails (n=50), following two disinfection protocols for the material used to manipulate snails. In one case, we cleaned all the material with bleach, water, and ethanol 70%, as previously reported to avoid carry-over contamination in molecular techniques (protocol Nº1) (Bonne et al., 2008). In the other, we cleaned the material only with ethanol 70% (protocol Nº2).

DNA isolation

DNA was isolated as described by Caron et al. with some modifications using Chelex-100® (Bio-Rad) chelating resin (Caron et al., 2011). Tubes containing the snails’ debris after snail crushing, miracidia of F. hepatica flukes were centrifuged at 13000 x g for 1 min and washed twice with 200 µl of distilled water to eliminate ethanol traces. The supernatant was discarded and 150 µl of 5% Chelex-100^® (Bio-Rad) was added. The mixture was vortexed three times for 30 s and incubated for 1 h at 56ºC and then for 30 min at 95ºC in water bath. The mixture was then centrifuged at 13000 x g for 7 min. The supernatant was collected and stored at -20ºC. DNA concentration and purity (260/280 wavelength ratio) were measured with a spectrophotometer (Thermo Scientific, NanoDrop 2000).

Amplification by PCR

PCR was performed using the specific primers FhCO1F: 5’-TAT GTT TTG ATT TTA CCC GGG-3’ and FhCO1R: 5’-ATG AGC AAC CAC AAA CCA TGT-3’, which amplify a 405-bp fragment in F. hepatica, and the primers LymF: 5’-TCC TAC TTG GAT AAC TGT GGC A-3’ and LymR: 5’-TTA CAA ACA TGG TAG GCA TAT C-3’, which amplify a 258-bp fragment in snails (Cucher et al., 2006; Duffy et al., 2009). DNA from F. hepatica flukes and Lymnaea viatrix were used as positive controls. To confirm that snails were not infected with F. hepatica, they were analyzed by microscopic exami­nation and tested with the PCR for F. hepatica reported by Cucher et al., 2006. DNA from free-living parasites and water were used as negative controls. The PCR reaction mixture consisted of Buffer 1X (PB-L, Argentina), 250 µM of each dNTP (PB-L), 1 µM of FhCO1 primers, 0.1 µM of Lym primers (Life Technologies, USA), 3 mM of MgCl2, 0.02 U of Taq DNA polymerase (PB-L) and 100 – 500 ng of DNA in a final volume of 25 μl. The amplification parameters were: 95ºC for 5 min, followed by 40 cycles of 94ºC for 30 s, 53ºC for 30 s, and 72ºC for 90 s, with a final step of extension at 72ºC for 5 min. PCR products were resolved by 1.5% agarose gel electrophoresis stained with GelRed (Biotium, USA). Amplified bands were viewed under a UV transilluminator and the image of the gel was captured using the FOTO/Analyst® Investigator/Eclipse System (Fotodyne, USA).

RESULTS

PCR setup

To set up the duplex PCR, it was necessary to prepare in-house reference standards in the same matrix as field samples (Burkardt, 2000; Victor et al., 1993). For that purpose, we measured the average amount of DNA obtained from one medium-sized snail (250 ng/µl) and prepared in-house standards with that amount of snail DNA and diffe­rent amounts of F. hepatica DNA. Then, to set up the optimal conditions for the duplex PCR, we evaluated different amplification reaction mixtures and cycling parameters. Parameters such as primer concentration and extension time were critical to obtain two defined bands of the expected sizes on a mix of 250 ng of L. viatrix DNA and 100 ng up to 1 ng of F. hepatica DNA (fig. 1).

We determined the analytical sensitivity of the duplex PCR by mixing 250 ng of snail DNA and serial dilutions of F. hepatica DNA. The analytical sensitivity for F. hepatica was 1 ng/µl. We analyzed a total of eight miracidia and measured the average DNA obtained from one miraci­dium, which was 2.5 ng/µl. Thus, we confirmed that this duplex PCR was able to detect one miracidium in one snail (fig. 2). The specificity of the set of primers used for F. hepati­ca detection was already tested by Cucher et al. (2006). Also, we performed the duplex PCR on DNA extracted from snails infected with free-living larvae observed in the analyzed snails and no bands were obtained (data not shown).

PCR on field-collected snails

When protocol Nº1 was used, F. hepatica was detected in two out of the 50 snails by both microscopic examination and PCR and only one sample presented no bands (table 1). When protocol Nº2 was used, all samples were positive for snail, and F. hepatica was detected in one out of the 50 snails by both microscopic examination and PCR. F. hepatica was detected in six out of the 50 snails only by duplex PCR.

DISCUSSION

The traditional diagnosis of F. hepatica infestation by microscopic examination is simple and affordable. However, it requires experienced laboratory technicians and has low sensitivity for early stages, especially if snails die before cercarial release (Kaplan et al., 1995; Kaplan and Reed, 1997). Therefore, sensitive methods for rapid and accurate identification of F. hepatica are needed for epidemiological surveys and infection control. Molecular diagnosis based on DNA detection by PCR is a promising tool to detect F. hepatica DNA in snails, since this technique is rapid and sensitive and no fresh samples are required (Alba et al., 2015, Caron et al., 2011, 2008; Cucher et al., 2006; Magalhães et al., 2004).

Some authors have attempted to detect F. hepatica in snails by PCR. However, the multiplex PCRs developed until now are based on repetitive regions of F. hepatica DNA, which generates several bands, or can detect the parasite in a specific snail host (Alba et al., 2015, Caron et al., 2011; Magalhães et al., 2004). Other authors have developed Real-Time PCRs protocols using specific probes, all of which are expensive and some of which only detect F. hepatica DNA, but not the snail’s DNA (Alasaad et al., 2011; Schweizer et al., 2007).

The duplex PCR developed in this work was able to detect F. hepatica in snails as two single and bright bands, with only two primer pairs in one step. The band corresponding to the snail works as an internal control that guarantees the presence of DNA and avoids false-negative results. Besides, the snail PCR amplifies a polymorphism located within the helix E10-1 of the variable region V2 of the 18S rRNA gene and if it is sequenced and aligned with GenBank sequences (Gen Bank accession numbers: AY057089, EU241866, EU728668, and EU241865) it can be used to identify the snail’s species (Duffy et al., 2009).

Interestingly, the snail band was detected in all the reaction mixtures and cycling parameters evaluated to set up the duplex PCR, while the F. hepatica band was only amplified after increasing the extension time and reducing the concentration of primers specific for the snail band. This is in agreement with previous reports that suggest performing these modifications in multiplex PCRs when long products are weak or absent (Henegariu and Heerema, 1997).

The detection limit was 1 ng of F. hepatica in the pre­sence of Lymnaea spp. DNA, which is optimal since it allows the identification of one miracidium (2.5 ng/µl) in one snail, which is the biological unit intended to detect (Kaplan and Reed, 1997).

Once the duplex PCR was set up, we evaluated how to process the snails considering the same sample was going to be analyzed in the parasitology and molecular biology laboratory. Right after collection, snails were crushed and analyzed under microscopic observation in the parasitology laboratory, and snails’ debris were collected in tubes with ethanol for conservation at -20°C, until DNA extraction.

During the parasitological analyses, snails are handled with stainless steel clamps and glass Petri dishes. In contrast, during molecular biology analyses, extreme care is taken to avoid false-positive results, such as the use of disposable material and the physical separation of reagents and materials before the PCR reaction. Thus, to obtain accurate results, and considering what was already reported by Bonne et al. (2008), we compared two disinfection protocols of the stainless steel clamps and glass Petri dishes.

The duplex PCR developed in this work showed results similar to those obtained by microscopic examination when protocol Nº1 was used. The application of a rigorous disinfection protocol to clean the material between snail co­llection was critical to achieve these results. When protocol Nº2 was used, only one snail was positive under micros­copic examination and PCR while six snails were positive only by duplex PCR.

Interestingly, some of the snails that were positive only by PCR were processed right after the only positive snail detected by microscopic examination and the amplicon obtained showed a subsequently decreased in the signal, suggesting carry-over contamination. Although we cannot confirm that the six positive results only by PCR achieved with protocol N°2 are false-positive results, we strongly re­co­mmend using protocol N°1 to clean the material used in the parasitology laboratory to handle snails previously to DNA extraction.

Previous studies have shown a higher level of detection of F. hepatica by PCR than by microscopic examination (Caron et al., 2011; Cucher et al., 2006; Kozak and We, 2010). Cucher et al. (2006) analyzed two samples from Corrientes and San Luis, Argentina. Snails were identified as L. columella and 17.5% of snails were positive for F. hepatica by microscopic observation while 51.3% were positive by PCR in samples from Corrientes. Snails were identified as L. viatrix and 2.9% of snails were positive for F. hepatica by microscopic observation while 61.8% were positive by PCR in samples from the province of San Luis (Cucher et al., 2006). Kozak and We (2010) evaluated the performance of a PCR assay for the detection of F. hepatica in Galba truncatula snails in four geographical areas of Eastern Poland and obtained an overall prevalence rate of 26.6%, which varied from 21% to 84% according to the region. However, in these reports the authors did not clearly state which measures were taken to avoid carry-over contamination.

With our duplex PCR, only one sample showed no bands. This may be due to the traces of bleach or ethanol present in the sample. Thus, we confirm the importance of the snail band, since, when this band is absent, the result cannot be taken into account. In conclusion, the duplex PCR deve­loped in this work considerably shortened the working time, decreased the number of false-positive results, can detect one miracidium, and can be performed by a technician with no experience in parasitology.

Although analysis of a greater number of samples is needed to validate this PCR, these results suggest that this duplex PCR is a promising tool to estimate the potential infection risk of ruminants in areas endemic for fascioliasis. The duplex PCR is very convenient because it is a sensitive technique that allows the detection of F. hepatica in snails, significantly shortens the working time, and has an accep­ta­ble cost.

Acknowledgments

This work was supported by INTA (grants Nº 1115054, 1281101, and 1281103).

We thank Vet. Fernando Raffo, Vet. Catalina Lauroua and Mr. Raul Cabrera for their assistance during field sampling.

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