Protozoa as Contaminants of Surface Water – Methods of Detection

Vladimir Ćirković1, Aleksandra Uzelac1, Ivana Klun1, Olgica Djurković-Djaković1

 

1 Centre of Excellence for Food- and Vector-borne Zoonoses, Institute for Medical Research, University of Belgrade, Dr Subotica 4, PO Box 39, 11129 Belgrade 102, Serbia; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it

 

Abstract

The role of Giardia spp., Cryptosporidium spp. and Toxoplasma gondii as etiological agents of waterborne outbreaks may be underestimated. One reason is that the standard water quality control methodology is focused on the detection of coliform bacteria and viruses. With the development of novel molecular methods, the detection of protozoa in water has become more rapid and accurate. In 2005, the EPA published a standard operating procedure for the detection of Cryptosporidium and Giardia, while a protocol for T. gondii is still being developed. Here we present the general methodology for water sampling, filtration and molecular detection of Cryptosporidium, Giardia and T. gondii.

Keywords: protozoa, molecular detection, contaminants, surface water.

Introduction

 

Protozoa are a diverse group of unicellular eukaryotes that belong to the kingdom of Protista. A large number of protozoa are ubiquitous free-living organisms, which can be found in surface water, soil and even inside macroorganisms. They can be beneficial to the host organism, as symbionts for instance, or harmful, as parasites. Among the tens of thousands species of protozoa, only about a hundred are considered pathogenic, including those classified as emerging or re-emerging (Woolhouse et al., 2005). According to the transmission route, parasitic protozoa can be vector-borne or food- and water-borne. Here we review the most important species that can be water-borne, such as Toxoplasma gondii, Cryptosporidium spp. and Giardia spp., and the current options for their detection in surface waters.

 

Clinical Significance of Water-borne Protozoa

T. gondii host range includes all warm-blooded species and even some cold-blooded ones. This parasite is infamous for its ability to be transmitted vertically, although it is only a minor mode of transmission. Congenital T. gondii infection can result in severe clinical symptoms like hydrocephalus, chorioretinitis or even death, whereas the infection is generally mild in immunocompetent hosts, who may or may not exhibit clinical symptoms at all. Immunocompromised patients (HIV+ patients and patients on immunosuppressive therapy) may develop severe manifestations including encephalitis. The parasite's life cycle includes three distinct life stages: the tachyzoite, the bradyzoite and oocysts, which are all infectious (Dubey, 2009). Following primary infection characterized by proliferating tachyzoites, infection converts into the latent (chronic) stage characterized by tissue cysts which predominantly develop in skeletal muscles and the brain, and may remain viable through the host's lifetime. If the host is a food animal, its muscles – meat – may be the source of infection for further (animal or human) hosts, which makes toxoplasmosis a foodborne infection. On the other hand, oocysts are the environmentally resistant stage, which contaminate the environment including water. This stage is the product of sexual reproduction in the intestines of definitive hosts, the Felidae family; shed in cat faeces, they sporulate in the environment and ultimately end up in water, soil or on plants meant for human and animal consumption.

The Cryptosporidium host range includes more than 155 animal species (Marshall et al., 1997). The parasite predominantly resides in the epithelial lining of the intestine and is transmitted by water contaminated with faecal matter. Cryptosporidium spp. generally cause diarrhoea as the major clinical symptom, however, infections can be lethal in immunocompromised individuals. Similar to T. gondii oocysts, Cryptosporidium spp. oocysts are environmentally resistant. In surface water, they can stay viable for 18 months. Oocysts are highly resistant to chlorination and UV irradiation, which allows them to remain infectious in treated drinking water.

Giardia spp., like the Cryptosporidium, are intestinal parasites transmitted mostly through faecal contaminated water or via the faeco-oral route due to poor hygiene. Giardia also has a wide host range and produces environmentally resistant cysts that also resist chlorination (Marshall et al., 1997). Clinical symptoms of infection are either entirely absent or mild and usually include diarrhoea and abdominal discomfort. The infection is usually self-limiting; however, in rare instances it may persist and have severe consequences for the host.

Another clinically important water-borne protozoan is Entamoeba histolytica (Morrissette et al., 2002), the etiological agent of amoebic dysentery or liver abscesses, but unlike the previously described organisms its life stages are not resistant to standard water disinfection treatments. E. histolytica is transmitted by heavily contaminated recreational water (or untreated drinking water) via cysts, or via the faeco-oral route. Symptoms are similar to those caused by Cryptosporidium and Giardia.

 

Contamination of Surface Water with Protozoa

Naturally occurring surface water as well as water in swimming pools may often be contaminated with parasitic protozoa. In contrast, subterranean water is usually free of parasitic protozoa, due in part to its physical and chemical properties, but largely to the fact that sources of pollution, which occur predominantly on the surface, have limited accessibility. Major polluters of surface and recreational waters can be focal and dispersed. Focal polluters are sewage pipelines and wastewater collection and treatment plants. Specific problems with urban sewage networks include substandard, leaky pipelines and the absence or inadequacies of sewage filters and wastewater collectors. As a consequence, industrial and organic waste can drain unprocessed into surface waters and aquifers used as drinking water sources. Dispersed polluters include agricultural soil and pastures. The runoff from these, due to rain, snow melt or irrigation, can disseminate parasitic protozoa and chemical contaminants into ground and surface water. Natural phenomena such as droughts and floods, which occur more frequently due to climate change, directly impact protozoa dissemination and induce changes in protozoa population structures in different bodies of water, which can have adverse consequences on human and animal health. During periods of drought, all available water sources, even those of poor or questionable purity, may have to be utilized for livestock, irrigation and household purposes. Livestock consumes a lot of water, making it impractical and/or costly to mechanically or chemically purify, or even just boil prior to use. Protozoa proliferate in the infected livestock and are eventually secreted in large numbers into the environment. Usage of contaminated water for irrigation of fruit and vegetables meant for raw consumption also poses a direct risk to human health. Examples include lettuce and berries, which can be hard to wash thoroughly without damage and therefore (oo)cysts may remain on their surfaces even after processing. There is a risk also with fruits and vegetables marketed and packaged as 'ready to eat' (marketed as "no wash needed") prior to consumption. Floods can lead to increased runoff from pastures, the mixing of ground with surface waters, or different bodies of water, including wastewater, all which in turn disseminates protozoa into possible drinking water sources.

 

Water-borne Epidemics

Over the last few decades, multiple geographically distinct epidemics ultimately associated with consumption of contaminated water have occurred. These epidemics have not only highlighted the significance of protozoa as waterborne pathogens and dramatically pointed out the shortcomings in drinking water treatment protocols, but also demonstrated that the impact parasitic protozoa can have on human health is severely underestimated. The greatest waterborne epidemic in the USA occurred in 1993 in Milwaukee, WI, and has been estimated to have involved over 400.000 people. It is speculated that oocysts of the causative agent, Cryptosporidium spp., entered the public water supply as a consequence of unusually increased runoff from pastures due to melting snow (Mac Kenzie et al., 1994). Only two years later, in 1995, the first documented epidemic due to T. gondii oocysts in the public water supply, occurred in British Columbia, Canada (Bowie et al., 1997). As the Wisconsin epidemic had already shown, this epidemic too highlighted the inadequacies of the established drinking water treatment methodology to remove and/or inactivate (oo)cysts. The small size of (oo)cysts allows them to pass through most commonly used filters, while their relative resistance to chlorination and UV irradiation ensures survival in drinking water. Epidemics of giardiasis occur periodically, although noticeably more often during the summer, due to the increase in recreational use of water – swimming in particular.

 

Methods for Detection of Protozoa in Water

In light of the above, continuous monitoring of parasitic protozoa populations in water sources destined for human/animal consumption or recreational use is required. For that purpose, efficient and precise detection methods are necessary. In 2005, the United States Environmental Protection Agency (EPA) published standard operating procedures (SOP) for the detection of Cryptosporidium spp. (SOP no. 1622) and combined detection of Cryptosporidium and Giardia (SOP no. 1623) in water. These SOPs include recommendations for water sampling, filtration and downstream analyses by microscopy and PCR. The methodology represented in the flowchart (Figure 1) is the approach recommended by the US EPA, as well as by most of the published literature on the subject of (oo)cysts and protozoa detection in water samples, and its steps and procedures are detailed below.

 

fig01
Figure 1: Methodology flow chart.

 

Water sampling

The minimal volume of water necessary for analysis of the presence of parasitic protozoa is 10 L, while there is no upper limit. Published reports include volumes of 600 L and even more, when analyzing drinking water (Medema et al., 2001; Ali et al., 2004; Karanis et al., 2006). Sampling is carried out by collecting from a depth of 20-30 cm below the surface (Hörman et al., 2004). It is recommended to avoid water layers close to the surface or sediment, as they can contain various biological materials such as plant matter and mud, which can block filters and/or interfere with downstream analyses.

 

Filtration

Filtration of the collected water sample is necessary to concentrate the (oo)cysts into a volume suitable for further processing, usually measured in ml. For this purpose, water can be filtered through nitrocellulose or polycarbonate membranes (Ali et al., 2004) or filter cartridges, designed especially for environmental water sampling (Envirocheck®, CUNO®) (Medema et al., 2001; Fournier et al., 2002). Filter membranes and cartridge filters are available with several different pore diameters. SOPs 1622/1623, recommend the usage of cartridge filters with pore diameters of 1 µm (Telliard et al., 2005). Relevant published literature also describes usage of filter membranes with pore diameters of 0.45 µm and 0.2 µm, which have been found to successfully retain Giardia cysts and T. gondii and Cryptosporidium oocysts. Following filtration, the samples should be cooled to a temperature between 1-10ºC and delivered to a laboratory equipped for protozoa detection within 96 h (Telliard et al., 2005).

 

Filter elution

To obtain a sample suitable for further testing, it is first necessary to elute all bound particles from the filter membrane or matrix. Elution is performed with buffered salt solutions which usually also contain mild detergents. Elution buffers are especially formulated for efficacy in (oo)cyst and protozoa detachment from the filter matrix or membrane without causing damage or affecting viability. Most common elution buffers contain chemicals which are easily available (EDTA, Tris) and can be made in the laboratory. Once the elution buffer is loaded into the cartridge filter or the filter membrane is immersed in the elution buffer, it is placed on a laboratory shaker to facilitate particle detachment and left for a period of time. Next, the elution buffer is collected and centrifuged to obtain the pellet which contains the (oo)cysts and protozoa as well as other particles collected during filtration. The pellet is then washed several times with washing buffers, which are usually low molarity salt solutions but without detergents. The pellet is finally considered pure and suitable for downstream analyses when the supernatant obtained after centrifugation is clear (Telliard et al., 2005).

 

Isolation of oocysts/cysts

There are two most commonly used approaches for (oo)cyst isolation. One approach is based on immunomagnetic separation (IMS), which requires the usage of specific antibodies which recognize and bind to the (oo)cyst surface, coupled to magnetic beads. The main advantage of this approach is its high specificity afforded by the use of monoclonal antibodies. Magnetic beads, which are in fact small, spherical particles which have been magnetized or made superparamagnetic, are a convenient way of isolating the (oo)cysts using a magnet. As a result, the sample obtained after IMS is highly pure and contains only (oo)cysts. The most commonly used magnetic beads are Dynabeads, which are highly versatile and can be easily coupled to a variety of monoclonal antibodies. Commercial kits for the isolation of Cryptosporidium oocysts and Giardia cysts by IMS are available, and in fact, a combination kit for the simultaneous detection of both is recommended by EPA's SOP 1623. However, there is no IMS kit for T. gondii oocysts on the market, because there is no commercially available suitable monoclonal antibody. However, successful coupling of in-house developed monoclonal anti-oocyst T. gondii antibodies to Dynabeads and their use in IMS has been demonstrated (Dumètre et al., 2005; 2007).

The other approach is based on particle separation by centrifugation through a concentration/density gradient of some suitable medium. This medium can be a chemical substance, such as sucrose, which is very commonly used, or it can consist of particles. Percoll is an example of a commercially available medium made of silica particles coated with polyvinylpyrrolidone (Medema et al., 2001; Lemarchand et al., 2003). The working principle of this approach for particle isolation is separation and collection by size. The entire process is carried out in a centrifuge tube. The sample pellet is usually added to the layer of medium with the lowest concentration/density, situated near the top of the centrifuge tube, while the most concentrated/dense layer is at the bottom of the centrifuge tube. During centrifugation, particles which make up the pellet will be dispersed into the medium and travel through the gradient until they reach layers which are impermeable to them. This approach is often applied for the isolation of T. gondii oocysts. While it can be very successful, one concern with the use of this approach is that it requires experience. It is not always easy to visually identify the layer of medium which contains the (oo)cysts, especially when the number of (oo)cysts in the pellet is low. In addition, the sample pellet may contain impurities, such as sediment particles, of similar size as (oo)cysts, making it hard to obtain a pure enough sample for downstream analyses.

 

Microscopy

Microscopy can serve as the endpoint analytical method, or as a waypoint analytical method. As an endpoint method, it can be used to determine the presence (and number) of (oo)cysts in the sample. As a waypoint method, it is used to check whether the methodology up to that point has been successful in isolating (oo)cysts. There are several protocols for specimen staining for microscopy, using dyes for examination by visible light and fluorophores for examination by fluorescence detection. A commonly used contrast stain is Lugol's solution, which is useful as the majority of protozoa and (oo)cysts take up the dye and appear yellowish-brown. However, correct identification of Lugol-stained (oo)cysts or protozoa requires considerable experience, therefore it is not ideal as an endpoint analysis for non-expert laboratories.

EPA SOPs recommend using immunofluorescence assays (IFA) instead (Telliard et al., 2005). Immunofluorescence is a staining method which relies on specific antibodies coupled to fluorophores. One obvious advantage is specificity, as only (oo)cysts will appear fluorescent on examination, whereas another one is the high intensity of brightness of the fluorophores, which allows even a few (oo)cysts to stand out, thereby increasing the chances of detection. EPA's SOP 1622 and 1623 both recommend the use of IFA as a waypoint method. In addition, the SOPs also recommend 4',6-diamidino-2-fenilindol (DAPI) or propidium iodide (PI) in combination with IFA. DAPI and PI are fluorescent nucleic acid stains and can help identify (oo)cysts which contain nucleic acids and are thus deemed viable. The viability of (oo)cysts may be an important piece of information, but significantly more important is determining that nucleic acids are indeed present in the (oo)cysts, which allows for further downstream analyses which utilize nucleic acids as samples, such as PCR or RFLP or sequencing. Another important advantage provided by the IFA method is that it is fully transferrable to most flow-cytometers. Flow cytometry is primarily used to identify and quantify cell populations, but the technique is actually based on counting different sized particles. It has been successfully applied to oocyst enumeration (Lemarchand et al., 2003). The major advantage of this technique is quick and precise quantification of (oo)cysts from much larger sample volumes than can be processed by microscopy.

 

Molecular detection

Molecular detection implies usage of techniques which are based on the polymerase chain reaction (PCR). PCR is used for amplifying specific sequences within the genome. The selection of the sequence to be amplified is of great importance, as genomes are composed of coding segments, such as genes (specifically exons), and non-coding segments, such as satellite sequences. The majority of genes are not specific enough to discriminate between species, let alone subspecies, using PCR alone. However, in combination with other downstream analyses, such as sequencing or even restriction fragment length polymorphism (RFLP), genes like small subunit ribosomal RNA (SSU rDNA), which are conserved across species, become highly useful for species and subspecies identification. Some satellite sequences can be highly unique to individuals, like "fingerprints", and are often used in forensics (Sambrook et al., 2001).

The first step for molecular detection is extraction of nucleic acids from the sample. The nucleic acids suitable for PCR techniques include both genomic DNA (gDNA) and RNA, usually messenger RNA (mRNA). There are many commercially available nucleic acid extraction kits, but classical extraction methods can also be highly successful. The advantage of most kits is that they avoid harsh chemicals, are specifically designed to simplify the extraction process, and provide uniform yields of nucleic acids. Most kits follow the same protocol of enzymatic digestion of cellular materials, binding of DNA/RNA to a silica based matrix with high molarity salt solutions, washing out of all unbound materials with ethanol solutions and finally, elution of the nucleic acids from the silica matrix with low molarity salt solutions. However, as environmental samples are highly heterogeneous and contain tough biological materials, they require the use of a specific kit for each type of sample. In the case of extraction kits recommended for (oo)cysts, enzymatic digestion is not sufficient, but has to be combined with mechanical lysis performed in a bead-beater instrument (Hill et al., 2015).

Once the nucleic acids have been obtained, they are used as templates for amplification in a variety of PCR based reactions. They are performed in the thermocycler, a thermo-block which can be rapidly heated and cooled based on a preset pattern of temperatures and units of time (min or sec). More advanced thermocyclers also include fluorescence units, which are equipped with an excitation source and several detectors for particular wavelengths. These are used for real time PCR and quantitative PCR (qPCR), because the reaction result is directly visualized and may be followed while the reaction is still ongoing. In contrast, conventional PCR does not allow for the visualization of the reaction product per se; for this, the gel electrophoresis technique is required (Sambrook, 2001). There are many variations of PCR: real-time PCR is mostly used for diagnostics, nested-PCR used in both diagnostics and detection, nested-PCR-RFLP which is nested PCR in combination with restriction endonuclease treatment, multiplex-PCR is used for amplifying multiple sequences simultaneously, sequencing-PCR is used as a preparatory reaction for Sanger sequencing. Commonly used PCR variations are shown in Table 1. The various types of PCR mentioned have been successfully used as stand-alone techniques or in concert for the detection and identification of Cryptosporidium, Giardia and T. gondii.

 

Table 1: Overview of PCR-based techniques used for detection in environmental samples.
tab01

 

Conclusions

Epidemics caused by waterborne pathogens, characterized by gastrointestinal problems, occur periodically in Serbia. The number of such illnesses increases during the summer months, which is concurrent with the swimming season and recreational use of water. In addition, the availability of fresh produce and fruit is increased. It has been documented that certain types of produce and fruit may be more likely to retain and harbour (oo)cysts, even after being washed. Increased exposure to contaminated water may explain the summer rise in gastrointestinal problems. When waterborne epidemics occur, the usual suspects include bacteria, such as Escherichia coli, Salmonella spp. and viruses, such as Enterovirus, Rotavirus, and Norovirus. These microorganisms are indicative of water contamination by human and/or animal waste. However, human and especially animal waste often contains large numbers of protozoa, which are generally neglected as the causes of epidemics. Bacteria and viruses predominantly cause acute and often severe infections, while diseases caused by protozoa tend to be milder, and do not cause mortality, in immunocompetent hosts at least. For these reasons protozoan infections may go unnoticed, undetected and underestimated. However, water examination and source attribution for outbreaks in Western Europe, Scandinavia and the USA has shown that protozoa like Giardia and Cryptosporidium are often present in surface waters and cause outbreaks thus directly impacting human health. These epidemics have demonstrated the need to analyse different water sources, of both surface and groundwater, for the presence and biodiversity of protozoa, in Serbia as well.

Standard operating procedures for the detection of Cryptosporidium and Giardia in water samples were published in 2005 by the US EPA. The methodology presented in this paper is based on the methodology recommended in these SOPs and supplemented by relevant literature in the field of protozoa detection in water. Presently, the Centre of Excellence for Food- and Vector-borne Zoonoses (IMR, UB) is in the process of introducing and implementing molecular detection and species identification of Cryptosporidium spp., Giardia spp. and T. gondii in samples from diverse Serbian water sources. This work will fill an important knowledge gap for both the environmental community and public health community.

 

Acknowledgements

This review paper was supported by the Ministry of Education, Science and Technological Development of the Republic of Serbia (project grant III 41019).

 

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