Detection of Giardia intestinalis in water samples collected from natural water reservoirs and wells in northern and north-eastern Poland using LAMP, real-time PCR and nested PCR

Giardia intestinalis is a protozoan parasite causing gastrointestinal diseases in humans and animals worldwide (Karanis & Ey 1998; Smith et al. 2007; Reynolds et al. 2008; Ryan & Cacciò 2013). It has been estimated that ingestion of as few as ten cysts can lead to the development of infection in humans and animals (Rendtorff 1954). The clinical manifestation of giardiasis includes severe diarrhoea, abdominal cramps, nausea, weight loss, and an acute phase which usually lasts a few weeks and which may develop into a chronic recurring disease (Wolfe 1992; Farthing 1996; Adam 2001; Lebwohl et al. 2003). The prevalence of Giardia infections in humans was previously estimated at 2‒5% in industrialised countries and 20‒30% in developing countries (Thompson & Monis 2004). However, regarding Europe, the epidemiology report from ECDPC in 2014 confirmed 17,278 giardiasis cases, reported by 23 countries in the EU/EEA, with the highest number of cases reported by Germany (n = 4,011), followed by the United Kingdom (n = 3,628), which accounted for 44% of cases (ECDPC 2015).

This parasite is transmitted by the faecal–oral route, through contaminated water and food (Karanis et al. 2007; Yoshida et al. 2007; Baldursson & Karanis 2011; Efstratiou et al. 2017). The presence of Giardia cysts in water is a problem throughout the world. They are resistant to unfavourable environmental conditions and can remain viable for almost two months at 0‒2 °C (deRegnier et al. 1989). To date, at least 905 outbreaks associated with waterborne transmission of protozoan parasites have been reported, of which G. intestinalis has been responsible for 40.6% over the last 100 years (Karanis et al. 2007), 35.2% in the period 2004‒2010 (Baldursson & Karanis 2011) and 37% in the period 2011‒2116 (Efstratiou et al. 2017). In Europe, a major waterborne outbreak of giardiasis occurred in Bergen, Norway in 2004 (Nygård et al. 2006). Surface water such as rivers and lakes may be contaminated through the discharge of untreated and treated sewage and runoff of manure (Karanis et al. 1998, 2007; Hunter & Thompson 2005; Baldursson & Karanis 2011; Efstratiou et al. 2017). In terms of sources of contamination of water and food, a particularly important role is played by various hosts acting as reservoirs of infection, including farm, household, and wild animals. For example, semi-aquatic species such as beavers deposit their faeces in water, and commensal rodents (house mice, rats, muskrats) contribute to soil/food contamination affecting both humans and animals. Some species, e.g., birds or insects, may also act as vectors facilitating the mechanical transmission of cysts (Bajer 2008).

The distribution of Giardia infections in humans in Poland (1‒9% in children; 3‒7% adults) is similar to that noted in other European countries (Bajer 2008). However, knowledge about the occurrence of this parasite in surface water in Poland is fragmentary. So far, only a few studies have shown its prevalence in various bodies of water in this country. Giardia spp. cysts, among others, were microscopically detected in 85% of water samples taken from 13 WTPs located in eastern Poland (Sroka et al. 2013). Evidence of environmental contamination also derives from studies reporting the presence of this protozoan in surface water reservoirs and fresh fruits in different parts of Poland (Szostakowska et al. 2005; Jędrzejewski & Majewska 2007; Polus & Kocwa-Haluch 2014; Adamska 2015).

Differentiation of genotypes cannot be done on the basis of parasite morphology, but requires molecular methods (Karanis & Ey 1998; Plutzer et al. 2008; Plutzer & Karanis 2009). The aim of the study was to estimate the occurrence of G. intestinalis, including genotypes pathogenic to humans, in water samples collected from surface water reservoirs and wells in northern and north-eastern Poland using three molecular detection methods, namely loop-mediated isothermal amplification (LAMP), real-time polymerase chain reaction (real-time PCR) and nested PCR, and to compare the usefulness of these methods for screening environmental samples for Giardia cysts.

Water samples were collected twice from the same sampling sites. One sample was taken between May and June (when the water temperature is relatively cool), the other between August and October (when the water is warmer).

Fifty-litre samples of surface water and water from wells were collected and filtered on site with the use of a portable filtration set consisting of clean 10-L polythene vessels (used for taking water from the source), a peristaltic pump head (Masterflex L/S Easy-Load II) attached to the handle of a cordless drill/driver (DeWalt DC980 Type 10), an Envirochek capsule membrane filter (Pall-Gelman Laboratory, USA), a flow meter (PTFE, Aalborg, USA), a manometer, and elastic pipes. Water from lakes, the pond and the lagoon was taken 1–3 m from the shore. If necessary, in order to remove larger particles (for example, leaves), surface water was first filtered with a sieve (pore size 1 mm).

Water was filtered according to US EPA Method 1623 (United States Environmental Protection Agency) with the Envirochek capsule filters. Filtration was performed at a flow rate of 0.7 L/min, until about 500 mL of water was left in the vessel. Then, 200 mL of PET buffer [0.01% Tween 80, 0.02% PPi, 0.03% EDTA] was added to wash the vessel walls and filtration was continued. Following filtration, the capsule filter was filled with 100 mL of PET buffer, transported to the laboratory and kept at 4°C.

Each water sample was filtered with changing the pipe section to a new one between the water sample and the filter to avoid contamination.

Following completion of the filtration procedure, the filter was left for 15 min in a vertical position, then inverted and left for 15 min in the inverted position. Then, the first fraction of washings was placed in a 225-mL conical tube; the filter was filled again with 100 mL of PET buffer and shaken in a horizontal position for 15 min using an automatic shaker (CAT S50, Germany). Then, the filter was rotated (180°) and shaken again for 15 min. The second fraction of washings was added to the conical tube containing the first fraction, mixed, and centrifuged for 30 min at 1,250 × g and 4°C without braking, using a Hermle Z383 K centrifuge (Labortechnik, Germany). Next, the supernatant was removed and the obtained pellet (≤0.25 mL) was suspended in 2 mL of PBS buffer [1% BSA, 0.05% Tween 20] (larger pellets were suspended in 4 mL of PBS buffer) and stored for further analysis.

Prior to DNA extraction, the material obtained from filters was frozen three times at −70°C and thawed at 30°C in a water bath to disrupt the cyst walls and improve the efficiency of DNA extraction, which was performed using a Sherlock AX kit (A&A Biotechnology, Gdynia, Poland) according to the manufacturer's instructions. All of the PCR templates were treated with an Anty-Inhibitor Kit (A&A Biotechnology) which removes polyphenolic PCR inhibitors using specific absorption particles, thereby removing factors that could interfere with the PCR. The PCR templates were stored at −20°C.

For specific detection of G. intestinalis DNA, nested PCR, real-time PCR and LAMP assays, developed by Hopkins et al. (1997), Guy et al. (2003) and Plutzer & Karanis (2009), respectively, were used in this study as described below.

Nested PCR was performed with the use of a set of the primers GIAF, GIAR (outer primers) and RH4, RH11 (inner primers) specific to the small subunit (SSU) rRNA gene of G. intestinalis developed by Hopkins et al. (1997). The amplification reaction mixture consisted of 12.5 μL of the standard and ready-to-use PCR mixture 2xPCR Mix Plus High GC (A&A Biotechnology) containing recombinant Taq polymerase, PCR buffer, magnesium chloride, nucleotides, stabilisers, and gel loading buffer, 0.25 μM of each primer (Metabion, Germany), and 3 μL of DNA in the first PCR and 1 μL of DNA in the second PCR, respectively, in a 25 μL reaction volume. Amplifications were performed with an initial polymerase activation step (10 min at 95°C), followed by 35 cycles of denaturation (45 s at 96°C), annealing of primers (30 s at 55°C for the outer and 5 s at 9°C for the inner PCR), strand extension (30 s at 72°C), and final extension (4 min at 72°C). Nested PCR reactions were performed using a GeneAmp PCR System 9700 Thermal Cycler (Applied Biosystems, USA). The PCR products (292 bp) were analysed using a GelDoc-It Imaging System (UVP, USA) following electrophoresis on a 2% gel agarose, which was stained with Midori Green DNA Stain (Nippon Genetics Europe GmbH, Germany).

Real-time PCR was performed with the use of the β-giardin P241 primer-TaqMan probe set specific to a fragment of the G. intestinalis β-giardin gene (Guy et al. 2003). The amplification reaction mixture consisted of 12.5 μL of Real-Time 2x HS-PCR Master Mix Probe (A&A Biotechnology), 300 nM of each primer (Metabion), 200 nM of the hydrolysis probe (Metabion), and 3 μL of template DNA in a 25 μL reaction volume. The amplification programme, consisting of initial denaturation (10 min at 95°C), 45 cycles of denaturation (15 s at 95°C), and annealing and elongation (1 min at 60°C), was performed in an Mx3005P thermocycler (Stratagene, USA). PCR products were analysed using MxPro QPCR Software. The cycle threshold (CT) value, determining the cycle number at which the reporter's fluorescence exceeded the threshold value, was recorded. A sample was considered positive if the CT value was <45.

The LAMP assay was performed using a Loopamp DNA amplification kit (Eiken Chemical, Tokyo, Japan) based a set of primers specific to the EF1-α-gene of G. intestinalis and thermal conditions described by Plutzer & Karanis (2009). The reaction mixture consisted of 12.5 μL of 2× reaction mix buffer, 40 pmol of each of the primers FIP and BIP, 20 pmol of each of the primers LF and LB, 5 pmol of each of the primers B3 and F3 (GENEWIZ SZ, Suzhou, China), 1 μL of Bst DNA polymerase (8 U), and 2 μL of the extracted DNA in a 25-μL reaction volume. LAMP reactions were performed in an Applied Biosystems 2720 Thermal Cycler (Thermo Fisher Scientific, USA). The amplified products were analysed using a WD-9413B gel imaging analysis system (Beijing Liuyi Biotechnology, China) following electrophoresis on agarose gel (Biowest Regular Agarose G-10, Gene Company) stained with ExRed nucleic acid electrophoresis dye (Beijing Zoman Biotechnology, China).

All PCR/LAMP experiments were performed including Giardia positive controls (genomic DNA extracted from trophozoites of a Giardia strain cultured axenically in the Department of Tropical Parasitology, Medical University of Gdańsk, Poland) to ensure the correct functioning of the reaction and negative controls (water template) to eliminate contamination of the PCR components.

All of the samples found to be negative were re-tested for the presence of PCR inhibitors by mixing 4 μL of DNA template and 1 μL of the Giardia positive control.

Real-time PCR was carried out on an Mx3005P thermocycler (Stratagene, USA) with the Giardia-specific β-giardin primer-probe sets P434 (P-1), designed based on the Portland 1 sequence of the G. lamblia β-giardin gene (assemblage A), and P434 (H3), designed based on the H3 sequence of the G. lamblia β-giardin gene (assemblage B), according to Guy et al. (2003, 2004). The amplification reaction mixture and programme were identical to those used for the β-giardin P241 primer-TaqMan probe set described above.

The final nested PCR products of the positive samples, after being cleaned with a Clean-Up kit (A&A Biotechnology), were sequenced. The products of the sequencing reaction, performed with the primers described by Hopkins et al. (1997), were cleaned with an ExTerminator kit (A&A Biotechnology) and subjected to analysis using an ABI PRISM 310 automated DNA sequencer (Applied Biosystems, USA). The results were analysed using ABI PRISM DNA Sequencing Analysis version 3.7 for the Windows NT platform (Applied Biosystems) and GeneStudio^TM Professional (GeneStudio, Inc., USA).

In three of the water samples (templates 1S2, 3S2 and 18 S2) positive results were obtained only with the LAMP assay. The remaining water samples, which were positive in LAMP, were also positive by at least one additional method: real-time PCR or nested PCR (Table 2).

Among the 13 different surface waters investigated, Giardia DNA was detected in three lakes (Choczewskie, Wysockie and Osowskie) and the Vistula Lagoon located in Pomerania province, and in two lakes (Jeziorak and Pozorty) and one small pond located in the Warmia-Masuria province (Table 2, Figure 1). Water samples collected from the remaining six water bodies, all lakes (Żarnowieckie, Dłużecko, Sitno and Dobre in Pomerania province; Rydzówka and Mamry in Warmia-Masuria province) tested negative for Giardia DNA (Table 2).

Among five wells examined, Giardia DNA was detected in four, that is, in five of ten water samples collected (Tables 1 and 2).

Among all water samples collected in the period between May and June, nine (60%) samples were positive for Giardia spp. Of the samples collected during this period, samples (four lakes, the pond and Vistula Lagoon) from six natural water bodies (n = 13) and from three wells (n = 5) were positive. In water samples from the same sites collected between August and October, Giardia DNA was amplified in six (40%) of them (Table 2).

In the case of one well (located in the village of Radosze), two lakes (Choczewskie and Jeziorak) and the Vistula Lagoon, a positive result for Giardia DNA amplification was noted in both water samples collected (S1 and S2) from the investigated sampling sites. For water samples taken from the remaining sampling sites, one of two collected samples was positive (Table 2).

Sequencing of nested PCR products was successful in six water samples. Comparison of the obtained sequences with the Giardia sequences available in the GenBank confirmed that the detected PCR products were fragments of the Giardia intestinalis SSU rRNA gene. Derived sequences are deposited in GenBank under accession numbers KY489978‒KY489983.

Giardia intestinalis is responsible for symptomatic giardiasis, which has been noted in about 200 million people worldwide (Carmena 2010). Water and food, among other routes of infection, are the most common transmission vehicles. To date, Giardia has been isolated worldwide from aquatic environments, including natural surface water reservoirs (rivers, lakes), treated drinking water supplies, bodies of recreational water (swimming pools and water parks) and wells (Karanis et al. 1996a, 1996b, 1996c, 1998). Concentrations of Giardia cysts are commonly reported in the range of 0.01–150 per litre; however, they may reach higher values in agricultural runoff and urban wastewater effluent. Cysts can survive for 2–3 months in an aquatic environment; their viability decreases with increases in temperature and at extremely low temperatures (Carmena 2010).

Detection of small numbers of organisms in water is difficult and requires very sensitive techniques, such as molecular methods which additionally enable genotyping of G. intestinalis isolates, which is impossible using microscopy. Many molecular assays, usually based on gene markers such as 18S rDNA, glutamate dehydrogenase, triose phosphate isomerase, β-giardin, and elongation factor 1α (EF1α), have been developed to detect Giardia isolates in clinical and environmental samples with high specificity and sensitivity. The methods include variants of PCR and nested PCR as well as the recently developed LAMP assay (Karanis & Ey 1998; Guy et al. 2003, 2004; Caccio et al. 2005; Plutzer & Karanis 2009).

In this report, we present the results of detecting Giardia DNA in concentrates from 50-L samples from several bodies of water located in northern and north-eastern Poland for the presence of G. intestinalis using three different molecular detection methods: LAMP, real-time PCR and nested PCR. Positive DNA amplification was noted in 42%, 33% and 25% of the water samples, respectively. Therefore, all three methods detected the presence of Giardia DNA in extracts from particles recovered from environmental water samples. However, from the point of view of higher sensitivity, LAMP has the advantage. The results of our findings confirmed that G. intestinalis is present in various water sources, including surface-water reservoirs and wells in Pomerania and Warmia-Masuria provinces, Poland. Moreover, the results of multiplex real-time PCR showed that the detected strains of G. intestinalis belong to assemblages A and B, with genotype B predominating. This indicates that water in the investigated area may be a source of giardiasis in humans, who use contaminated water bodies for recreational activities and water from contaminated surface wells for gardening.

G. intestinalis has been found in various water reservoirs worldwide. In Poland, there have been very few studies concerning detection of G. intestinalis in water sources. Giardia spp. cysts, among others, were found using microscopic methods in 85% of water samples taken from 13 sewers located in eastern Poland (Sroka et al. 2013). The presence of G. duodenalis was also confirmed in recreational water reservoirs in north-eastern Poland with the use of direct fluorescence assays (46%) and molecular methods (48%) (Sroka et al. 2015). Another study by Bajer et al. (2012), performed using IFA and nested PCR assays, showed the frequent occurrence (61%) of this parasite in surface water in Poland. Moreover, Polus & Kocwa-Haluch (2014) found Giardia DNA in four of 17 water bodies investigated in the vicinity of Cracow (southern Poland). The presence of this protozoan has also been confirmed using molecular assays in several water bodies used for recreational activities in north-western Poland (Adamska 2015). Earlier studies performed in the Tri-City area (northern Poland) using the FISH/IF method also demonstrated the presence of Giardia cysts in three of seven examined lakes (Szostakowska et al. 2005).

It is known that genotypes A and B of G. intestinalis are responsible for infections in humans (Karanis & Ey 1998). Genotype A is associated with a mixture of human and animal isolates, while B tends to be predominantly associated with human isolates (Carmena 2010). Genotyping performed in our studies using real-time PCR showed that most of the investigated samples were contaminated with G. intestinalis assemblage B. These results are in accord with other studies performed in Poland showing the occurrence of both zoonotic assemblages in various water sources, with a higher frequency of genotype B (Adamska 2015; Sroka et al. 2015).

We found more positive water samples among those collected during spring and early summer as opposed to late summer (nine of 16) and autumn (six of 16). These results are in accord with the findings of Sroka et al. (2015), who detected Giardia cysts in samples collected from recreational water reservoirs in the Warmia-Masuria province in spring with a frequency approximately three times higher than that in autumn. This may result from changes in the prevalence of infection in hosts which excrete cysts of the parasite and contaminate the environment, as well as from the fact that the viability of Giardia cysts decreases with increasing water temperatures.

The source of contamination of water may vary, but it is always connected with the excretion of cysts by infected hosts (humans or animals). The prevalence of Giardia infections in the Polish population ranges between 1 and 8.8% in healthy children and 3.1 and 6.5% in healthy adults (Bajer 2008; Korzeniewski et al. 2014, 2016). Giardia spp. infections appear to be rare in farm animals in Poland (cattle 2.2‒14%; sheep 1.3%) and more prevalent in wildlife. This parasite has been found in a range of host species, being especially common in rodents (including species of semi-aquatic rodents, e.g., European beavers and muskrats, with frequencies of 7.7% and 87%, respectively) and wolves (20‒46%, depending on the method of detection), and less prevalent in other species such as European bison, roe deer and red deer (Karanis et al. 1996b, 1996c; Paziewska et al. 2007; Bajer 2008). Giardia cysts were also detected in house animals: in 6‒36% of dogs, and in a small percentage of investigated cats (Bajer 2008; Jaros et al. 2011).

The source of contamination of water investigated in our study may differ depending on the localisation and character of particular sampling sites. Choczewskie is a very clean, flow-through lake situated in the Choczewo-Salino Protected Landscape Area (Pomerania province), a popular site for tourism and recreation. Here, we detected G. intestinalis assemblage B, which may derive mainly from wildlife; however, a human origin cannot be excluded. The lakes Wysockie and Osowskie are small ribbon lakes. Low quality water from Wysockie (Gdańsk district), showing progressed eutrophication induced by the discharge of sewage into the aquatic system, is used mainly for factories and irrigation of agricultural areas. Osowskie, situated on the border of the Kashubian Lake District, is cleaner that Wysockie, but also polluted with waste coming from local farms as well as from a storm water drain. In these lakes, the occurrence of G. intestinalis assemblages A and B may have a human or mixed host origin. Jeziorak, a large ribbon lake, located in Warmia-Masuria province, has a rich lakeside and is surrounded by the marvellous forests of Iława Lake District Landscape Park. In this lake, we found DNA of G. intestinalis assemblage B, the source of which may be both human (i.e., waste may derive from certain yachts whose owners avoid treatment stations in marinas) and animal (wildlife from the surrounding landscape park and domestic animals from local homes). In contrast to Jeziorak, Pozorty is a small lake, surrounded by forests and fields, where the contamination most probably originates from wildlife and domestic animals. The Vistula Lagoon has poor quality water due to waste from sewage treatment plants and several rivers flowing into the lagoon. Therefore, the contamination with Giardia is probably of human origin. We also found Giardia DNA in wells located on private property (kitchen gardens/yards) in small poor villages in Warmia-Masuria province. The homes are located in an agricultural area, surrounded by forests and fields, and inhabited and/or visited by both domestic animals (dogs, cats) and wildlife. Although it is difficult to indicate the exact source of contamination in this case, the results are similar to the findings of other researchers who also detected Giardia and/or other parasites in these wells (Sroka et al. 2006; Alam et al. 2014).

The present study supplements previous information for Poland on the distribution of Giardia in surface waters, including five lakes, one pond and a lagoon in northern and north-eastern Poland. It provides a comparison of molecular detection methods (LAMP, RT-PCR and nPCR) for Giardia in concentrates recovered from filtering water. Management of sources contamination and investigations of possible giardiasis cases in the affected areas in association with control measures may effectively support control programme measures against transmission routes of Giardia in humans and animals in Poland.

In the present study, we detected DNA of G. intestinalis in surface water samples taken from lakes, a pond and a lagoon, as well as from wells located in Pomerania and Warmia-Masuria provinces, Poland. These findings indicate a potential risk for people using contaminated water. Moreover, we have demonstrated that the LAMP assay was the most sensitive of the three molecular detection methods used for screening of the environment for Giardia species.

We acknowledge the funding support from the MN 01-0188/08/772 project and One Thousand Talents Plan of the Chinese Government (No. WQ2013630172). The manuscript does not contain clinical studies or patient data. The authors declare that they have no conflict of interest.