2.3. Sample processing

The water samples were shaken manually for around 5 s before filtering through polycarbonate membrane filters (diameter 47 mm; pore size 2.5 μm; REATRECK FILTER Ltd., Obninsk-3, Kaluga Region, Russian Federation). When the filter clogged, it was removed and refrigerated, and a new filter was used to continue the filtration. For each sample between two and 14 filters were required depending on the amount of particulate material in the sample. When the sample had been filtered, each membrane filter was split into two equal parts using a sterile scalpel.

The sediment samples were transferred to 15 mL centrifuge tubes and centrifuged for 3 min at 1500 rpm (430 g) and the supernatant removed. The sediment material was combined and stored refrigerated (+4 °C) in plastic tubes before transporting to the Norwegian University of Life Sciences for further analysis.

For 23 of the 24 water samples, one half of each filter for that sample was placed on a microscope slide and fixed in 96% ethanol, covering the whole filter for 10 min. The slide was flame-dried for 3–4 s, then stained using the standard mZN technique (e.g., Garcia et al., 1983; Casemore, 1991), with carbol fuschin stain, decolourisation with 3% hydrochloric acid, and Brilliant Green counterstain. The samples were then screened by light microscopy at x1600 (objective 100×, eyepiece 16×) using immersion oil and oocysts identified by their characteristic pink colouration and size (3–5 μm diameter) against a green background (see Fig. 2). The number of oocysts seen per sample was registered.

Cryptosporidium sp. oocyst (indicated by red arrow) on filter surface (stained by mZN) among other debris. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

For one sample, one half of each filter used for concentrating the sample was stained with Lugol solution on a microscope slide, at the orientation that the filter collection side was upward, and Lugol solution added to cover the filter. The samples were then screened by light microscopy and Giardia cysts identified by morphological criteria. The number of cysts seen per sample was registered.

For two of the 29 samples, one half of each filter used for that sample was placed on a microscope slide, at the orientation that the filter collection side was upward, and fixed by methanol solution added to cover the filter. The samples were then examined by immunofluorescent antibody test (IFAT), with the fixed sampled stained with commercially available (AquaGlo, Waterborne Inc., New Orleans, USA) monoclonal antibodies against Cryptosporidium oocyst walls and Giardia cyst walls, labelled with fluorescein isothiocyanate (FITC) by incubation at 37 °C for 30 min. Following incubation, the samples were stained with 4′,6-diamidino-2-phenylindole (DAPI), rinsed and then anti-fade mounting medium (1,4-diazabicyclo [2.2.2] octane; DABCO) added, before screening by fluorescence microscopy at x250 (and x400, if required) using appropriate filter blocks for visualising FITC and DAPI. Cryptosporidium oocysts and Giardia cysts were identified by characteristic fluorescent staining and morphological criteria. The number of parasites seen per sample was registered.

For 11 samples, chosen on the basis of mZN results (eight had been found to be positive for Cryptosporidium oocysts by mZN), one half of each filter was cut into small pieces using scissors or scalpel, and the pieces added into kit tubes for DNA extraction (BioFACT Genomic DNA Prep Kit, SmartScience Co. Ltd., Bangkok, Thailand), with between four and eight pieces of filter snippets per tube. DNA was extracted according to the manufacturer's protocol. The eluted DNA was frozen at −20 °C before transportation to the Norwegian University of Life Sciences for analysis by qPCR.

For each DNA eluate, qPCR was conducted for detection of DNA of Cryptosporidium spp., Toxoplasma gondii, Echinococcus multilocularis, and Cyclospora cayetanensis using the protocols described by Temesgen et al. (2022). This consists of one multiplex PCR using 2 μL of template (for T. gondii, E. multilocularis, and C. cayetanensis; Temesen et al., 2019) and a separate simplex PCR for Cryptosporidium using 5 μL template and the protocol of Elwin et al. (2022).

The qPCR was run in using a Stratagene AriaMxReal-Time PCR System (Agilent Technologies, Inc., Santa Clara, CA, US) with Agilent AriaSoftware v1.5., with all samples run in two technical replicates of duplicate samples, and using appropriate positive and negative controls in each run.

A mixed aliquot of approximately 5 g of each of the two sediment samples were resuspended in water and transferred to L10 tubes, together with the necessary buffers and beads for immunomagnetic separation (IMS) of Cryptosporidium oocysts and Giardia cysts using the Dynabeads Cryptosporidium/Giardia Combo Kit (Idexx, UK) according to the manufacturer's instructions. After mixing for 1 h, and following subsequent dissociation of the beads and potential parasites by vigorous shaking in 50 μL of 0.1 M hydrochloric acid, the suspension was added to a welled slide, neutralised by addition of 5 μL of 1 M sodium hydroxide and air-dried. The dried concentrate was fixed in methanol, then stained with AquaGlo as described above in Section 2.3.4, and examined as previously described for IFAT by fluorescence microscopy.