Key words

1 Introduction

Toxoplasma gondii is a zoonotic parasitic protozoan, capable of infecting humans and other homeothermic animals. Several forms of transmission occur in the toxoplasmosis cycle, among them, waterborne transmission emerges as an important transmission route capable of causing large-scale outbreaks [1, 2].

Felids, especially domestic cats, are the T. gondii definitive hosts, so in them parasite sexual development occurs, this infection culminates in oocyst production, which are shed through feces into the environment. Under temperature and humidity suitable conditions, oocysts become infectious and can be carried to watersheds [3]. T. gondii oocysts are highly resistant to environmental conditions and can remain viable for months to years in the environment; this evolutionary form is also resistant to the main disinfectants widely used, including those based on chlorine widely used in water treatment [4, 5].

Among waterborne diseases, several etiologies are known [6]. Among protozoa, Cryptosporidium , Giardia , and T. gondii are the main etiologies in protozoa waterborne outbreaks [7, 8]. Toxoplasmosis outbreaks have historically been described as restricted and commonly related to community food transmission mainly through the ingestion of tissue cysts ; however, since 2000, waterborne transmission has been described more frequently [1,2,3,4,5,6,7,8,9].

The diagnostic of T. gondii in water matrices, mainly in raw and treated water, is essential for the characterization of occurrence and distribution of this protozoa in different locations, from this knowledge parameters based in risk assessment can be estimated to provide a better control of toxoplasmosis dissemination [10]. In epidemic situations, specifically when a water source is considered suspected in an outbreak, a positive outcome in a water test can confirm the evaluated source as the outbreak cause. Nevertheless, T. gondii oocyst detection in water samples is complex as the known methods lack sensitivity and specificity [10, 11].

The main factor that negatively impact the diagnostic techniques applied to water testing is the diversity of the physical-chemical characteristics of water in the environment, especially turbidity; this provide a great variation in the performance of the methods used for diagnosis [12, 13].

Different protocols for T. gondii diagnostic in water samples are known, from simple techniques whose are mainly applied to direct diagnostic in fecal samples from definitive hosts to techniques more adapted to inherent necessities of methods applied to environmental investigations [14]. Between the most modern techniques stand out the immunomagnetic separation efficiency, the purification of concentrated samples, and the diagnostic/quantification ability of qPCR; however, there is still no commercial availability of kits for wide use in routine diagnostic [15, 16, 27]. Specifically with respect to T. gondii, morphological identification methods are not able to distinguish T. gondii oocysts from at least four other coccid species (Hammondia hammondi, H. heydorni, Neospora caninum, and Besnoitia); therefore, the simple direct transposition of methods routinely used in hosts may not be suitable for environmental matrices [3, 17].

Considering the absence of commercial kits that include these new diagnostic advances, the methods used as a routine to investigate the presence of T. gondii oocysts in water samples still consist of sample concentration by filtration, elution with surfactant solution (Tween® 80 0.1%), later centrifugal concentration of the eluted material, with or without a fluctuation purification step in a dense solution (sucrose, cesium chloride) [18,19,20]. For visualization and identification of parasitic forms by microscopy, UV light microscopy is superior when compared with brightfield microscopy since coccid oocysts present a characteristic autofluorescence when exposed to this wavelength [20, 21].

This technique, based on concentration by filtration, surfactant elution and microscopic visualization, was adapted from methodologies applied to diagnosis and monitoring of Cryptosporidium and Giardia . The different methodologies applied to T. gondii oocyst diagnosis and monitoring in water samples lacks efficiency, especially when compared to the reported efficiencies of methods applied to other protozoa such as Cryptosporidium and Giardia , for these ones worldwide standardized and validated methods are available [22]. Filter cartridges eluted by agitation indicated for Cryptosporidium and Giardia , when applied for concentration of T. gondii oocysts in high-turbidity samples, present a recovery efficiency 10 times lower than the recommended [10].

Since the popularization of molecular techniques is applied to diagnosis , detection by PCR started to be commonly applied as it has a greater sensitivity and specificity and can differentiate T. gondii from other coccids. However, the relative resilience of oocysts to methods generally applied to cell disruption, in addition to the known presence of PCR inhibitors in environmental and fecal samples, represents a challenge for obtaining pure DNA [23, 24]. In order to overcome the occurrence of inhibition on genetic material amplifications, studies have demonstrated a greater robustness of the loop-mediated isothermal amplification (LAMP) technique, which is not inhibited by the main inhibitors present in the environment; however, it is not yet a widely known and used technique like PCR [25, 26].

Therefore, to avoid occurrence of false negatives due to analytical inadequacy, an extensive method validation must be performed, as well as an internal amplification control (IAC) must be employed in the PCR . Methods used to monitor the presence of pathogens in water and food for human consumption must be thoroughly tested, and the extent of the influence of pre-analytical factors must be known since these data provide greater reliability of the obtained results [22].

Molecular diagnostic techniques such as conventional PCR and LAMP do not allow oocyst quantification within a sample, so techniques that allow quantification such as qPCR must be validated for a better understanding of the occurrence and contamination dimension [10, 27,28,29].

2 Materials

  1. 1.

    Distilled water

  2. 2.

    Tween 80 (Polysorbate 80) 0.1% (V/V).

  3. 3.

    Vacuum pump.

  4. 4.

    Silicone tubes.

  5. 5.

    Kitassate 4 L.

  6. 6.

    Filtration system for 47 mm diameter membrane.

  7. 7.

    47 mm Mixed cellulose esters (MCE) membranes, maximum porosity of 5 μm.

  8. 8.

    Disposable petri dishes.

  9. 9.

    Calibrated loop.

  10. 10.

    Disposable Pasteur pipettes.

  11. 11.

    Conical tubes for centrifugation (50 mL).

  12. 12.

    Centrifuge with rotor for conical tubes.

  13. 13.

    Crystal sugar.

  14. 14.

    Densimeter.

  15. 15.

    Phenol.

  16. 16.

    Aqueous sucrose solution (sp.g. 1208 g/L).

  17. 17.

    Microtubes.

  18. 18.

    Brightfield microscope.

    Optional: Phase-contrast, differential interference contrast (DIC), UV light microscope (330–380 nm excitation and 400 nm barrier).

  19. 19.

    DNA Extraction kit (including all materials required from the manufacturer).

  20. 20.

    General PCR reagents and equipment.

  21. 21.

    General DNA electrophoresis reagents and equipment.

3 Methods

3.1 Indication

This protocol is indicated for the concentration, purification , and detection of T. gondii oocysts in raw, treated, spring, and ground water samples.

3.2 Sampling

When the water is piped, sampling can occur directly from the tap. If it is not, sampling can occur directly in the watershed or fountain, in this case the sampler must be cautious to not revolve the soil/sediment. The minimum water volume to be processed by this technique is described below:

  1. 1.

    Raw water: at least 3 L.

  2. 2.

    Treated water: 100 L.

  3. 3.

    Spring and groundwater: 100 L.

3.3 Concentration

A high oocyst dilution is expected in water matrices, this can drive to a low oocyst concentration on collected sample, so a concentration step aims to concentrate the oocysts to a low volume for detection ; as there is not a concentration technique able to concentrate only T. gondii oocysts , it is important to take into account that other samples constituents (e.g., organic/inorganic matter, soil, etc.) will be also concentrated (see Note 1).

3.4 Filtration

  1. 1.

    Using the silicone tubes, plug the vacuum pump to the kitassate.

  2. 2.

    Connect the filter system to the kitassate, ensure that the system is well placed to not lose vacuum pressure during filtration.

  3. 3.

    Place the membrane in the designed space of the filter system and lock with the filter system clamp.

  4. 4.

    Turn on the vacuum pump.

  5. 5.

    Rinse the whole filter system with Tween® 80 (0.1%).

  6. 6.

    Add sample in the filter system cup and continue adding the sample during the filtration.

    1. (a)

      Whenever the filtration speed dramatically decreases, change the membrane, as the filter membrane pores have saturated (the frequency of the membrane change will depend on the turbidity of the water sample).

    2. (b)

      If the membrane has saturated and some sample is still in the filtration cup, use a disposable pipette to remove the sample until the membrane appears dry.

    3. (c)

      Be careful with the vacuum pump temperature ; long period filtration can cause a pump overheat.

    4. (d)

      Be careful with the filtrate volume and kitassate capacity; whenever the kitassate reaches the capacity, remove the vacuum pressure, remove the filtration system and drain out the kitassate.

  7. 7.

    Place used membranes, with the concentrated matter, in a disposable petri dish and add Tween 80 0.1% until it is covered.

  1. (a)

    If membrane filtration and membrane elution are not performed in the same place, the membranes can be packaged in a plastic bag with Tween 80 0.1% and kept under refrigeration (4 °C).

3.5 Membrane Elution and Centrifuge Concentration

  1. 1.

    With the loop part of a calibrated loop, perform a smooth membrane scraping in different directions during 20 min in each membrane.

    1. (a)

      If membranes are placed in a plastic bag, carefully remove the membrane and place them in a disposable petri dish with Tween 80 0.1% for scraping, and save the liquid.

  2. 2.

    After scrapping, transfer the Tween 80 0.1% solution from the petri dish to a centrifuge conical tube.

    1. (a)

      If membranes are placed in a plastic bag with Tween 80 0.1%, also transfer the plastic bag liquid to the centrifuge conical tube.

  3. 3.

    Centrifuge the tubes (2100 × g/10 min).

  4. 4.

    Carefully remove the supernatant using a disposable Pasteur pipette. Leave a minimum quantity of supernatant for pellet resuspension. Resuspend the pellet using the Pasteur pipette.

    1. (a)

      If more than one tube is necessary to fit all the liquid generated in the elution, transfer all the resuspended pellets from the same sample to a single conical tube.

  5. 5.

    Centrifuge the tubes (2100 × g/10 min).

  6. 6.

    Carefully remove the supernatant using a disposable Pasteur pipette. Leave a minimum quantity of supernatant for pellet resuspension.

  7. 7.

    When the final sediment is greater than 500 μL, purification must be performed before detection .

3.6 Purification

During the membrane elution and centrifuge–concentration step, various organisms and environmental compounds were concentrated in addition to the oocysts , so when a high pellet volume is obtained in concentration, the purification step is necessary to remove as many components as possible that could interfere with the detection phase.

  1. 1.

    Resuspend the concentrated sample (0.5–5 mL) in 40 mL of sucrose solution (g.sp. = 1.208 g/mL) in a 50-mL conical tube.

    1. (a)

      T. gondii oocyst density is 1.11 ~ 1.14 g/mL.

  2. 2.

    Centrifuge at 1250 × g for 10 min.

  3. 3.

    Collect 5 mL of the superficial meniscus using a Pasteur pipette, and transfer it to a new 50-mL conical centrifuge tube.

  4. 4.

    Add 40 mL of distilled water and mix vigorously.

  5. 5.

    Centrifuge at 2100 × g for 10 min.

  6. 6.

    Carefully discard the supernatant using a Pasteur pipette and resuspend the formed sediment with 0.5–1.5 mL of distilled water (uses the sufficient volume necessary to resuspend the sediment and form a homogeneous appearance mixture).

  7. 7.

    Place the purified material in a sterile microtube and store under refrigeration (4 °C) until detection .

    1. (a)

      Detection steps must be performed within 48 h.

3.7 Detection

In the detection step, there is a direct parasite diagnostic, through evaluation of microscopic morphological structure, or through PCR DNA amplification.

3.7.1 Microscopic Detection (See Note 2)

  1. 1.

    Wet-mount 20 μL of the concentrate/purified on slide and coverslip.

  2. 2.

    Screen the slide in brightfield microscopy, phase contrast or UV light microscopy with 330–380 nm excitation and 400 nm barrier in the 40× objective.

  3. 3.

    When positive, the sample will present oocysts of approximately 12 μm, when not sporulated, characterized by a spherical shape with a modulated interior, and when sporulated, a spherical to elliptical shape containing two sporocysts with four sporozoites inside (see Note 3).

3.7.2 PCR Detection

3.7.2.1 DNA Extraction

To obtain more accurate results in the polymerase chain reaction (PCR) , it is suggested to extract DNA by means of commercial kits. The most efficient DNA extraction and purification are those indicated for extracting nucleic acids from faces and environmental samples (e.g., soil, water, soil, and plants; water, for drinking water, raw water).

A method validation should be carried out to assure that the kit protocol is able to extract DNA from T. gondii oocysts .

3.7.2.2 PCR Targeting the rep529

A PCR targeting the non-codified 529 bp repeated sequence from T. gondii genome based on primers tox4 and tox5 described by Homan et al. (2000) is indicated.

  1. 1.

    Assembly reaction components (Table 1) in an identified PCR microtube (200 μL) or in each PCR microplate well.

  2. 2.

    Allocate microtubes in thermocycler. Check reagents’ manufacturer’s instructions regarding PCR cycle. A cycle example is described in Table 2.

Table 1 Components, concentrations, and volumes for assembly of PCR targeting the 529 bp repeated fragment of Toxoplasma gondii genome
Full size table
Table 2 Example of cycle conditions for a PCR targeting the 529 bp repeated fragment of Toxoplasma gondii genome
Full size table

After PCR amplification, run an electrophoresis for DNA detection as follows:

  1. 1.

    Prepare 1.5% (w/v) agarose gel with TBE or TAE buffers.

  2. 2.

    Run the electrophoresis with DNA dye and loading buffer of preference following the manufacturer’s instructions.

  3. 3.

    Expose the gel to indicated wavelength of the DNA dye used, a sample is considered positive when a DNA band of 529 bp is observed (see Note 3).

4 Notes

  1. 1.

    High turbidity levels negatively affect technique efficiency.

  2. 2.

    T. gondii oocysts are microscopically indistinguishable from those of Neospora and Hammondia genus. Oocysts of coccids when exposed to UV light emit fluorescence in blue color, facilitating their visualization in complex samples. Due to the difficulty of microscopy detection mainly caused by low final volume of the sample analyzed, presence of confounding structures and the non-differentiation between Neospora and Hammondia and T. gondii oocysts , concomitant use of molecular methods is recommended.

  3. 3.

    In outbreak situations, laboratory tests results should be evaluated together with epidemiological analysis, as a negative test does not exclude the possibility of T. gondii oocysts presence, so T. gondii diagnostic techniques applied to environmental samples can present low sensitivity among other causes due to the difficulty in collecting and/or concentrating large volumes. Time elapsed between the first clinical signs, case notification, and sample collection should always be accounted in outbreak situations.