Formol

DNA was extracted using a QIAmp DNA stool mini kit (QIAGEN, Hilden, Germany), following the manufacturer’s protocol. Extracts were thereafter stored at −20 °C. A part of the stool samples was preserved in 5% formalin to perform the microscopic analysis. PCR investigations were carried out on site. In addition, extracts and formalin samples were sent to the Bernhard–Nocht Institute for Tropical Medicine (BNITM, Hamburg, HH, Germany). During the BNITM microscopy, control examinations and further analysis were performed. Airfreight requirements were fulfilled, the cooling chain was not interrupted (based on the temperature control documentation), and transport was done by a specialized company (World Courier, Frankfurt, HE, Germany).
In-house real-time multiplex PCRs for protozoan and helminthic parasites targeting Entamoeba histolytica, Giardia intestinalis, Cryptosporidium spp., and Cyclospora cayetanensis, as well as Necator americanus, Strongyloides stercoralis, Ascaris lumbricoides, Ancylostoma spp., Trichuris trichiura, Schistosoma spp., Enterobius vermicularis, Taenia saginata, Taenia solium, and Hymenolepis nana were performed as described before [3 (link)]. Further, enteroinvasive bacterial pathogens like Campylobacter jejuni, Salmonella spp., Shigella ssp./enteroinvasive E. coli (EIEC), and Yersinia spp. were assessed by in-house real-time PCR [4 (link)]. To identify diarrheagenic E. coli infections, Rida Gene RT-PCR assays for enterohemorrhagic Escherichia coli (EHEC), enteropathogenic E. coli (EPEC), enteroinvasive E. coli (EIEC), enterotoxin-producing E. coli (ETEC), enteroinvasive E. coli (EIEC), and enteroaggregative E. coli (EAEC) (R-Biopharm AG, Darmstadt, HE, Germany) were used, as described before [5 (link)]. Furthermore, a species-specific PCR for Tropheryma whipplei [6 (link)] was applied [7 (link)]. The DNA of phocid herpesvirus was included as an internal control for the in-house PCRs [8 (link),9 (link)].
In all RT-PCRs runs, positive and negative controls were included: As positive controls, synthetically designed target sequences linked by EcoR1 endonuclease restriction sites and inserted into pEX-A128 vector backbones were used (Eurofins Scientific SE). Negative controls included PCT water samples that had undergone the whole nucleic acid extraction process to exclude sample contamination. All primers and probes of the in-house PCRs were purchased from Eurofins, Hamburg, HH, Germany. The assays were performed on a multi-channel RotorGene Q Cycler (Qiagen, Hilden, HE, Germany).
In addition, light microscopy of all stool samples by direct saline and/or iodine mounts and following a formol-ethyl acetate concentration was performed.

At post-mortem, each brain was divided into two parts by a sagittal paramedian cut. The smaller portion was immediately frozen and stored at −20 °C for Western blotting. The remaining part was immersed and fixed in 10% formol saline for 4 d. The brains were trimmed at standard coronal levels, decontaminated with formic acid for 1 h, and embedded in paraffin. Sections (6 μm thick each) were cut for haematoxylin and eosin staining, immunohistochemistry, and PET blot, randomly mixed and coded for pathological assessment.
For the construction of lesion profiles, vacuolar changes were scored in nine grey-matter areas of the brain on haematoxylin and eosin-stained sections, as described by H. Fraser and A. G. Dickinson [49 (link)]. Vacuolation scores are derived from at least six individual voles per group and from three individual mice per group, and are reported as means ± standard error of the mean.
For PrP immunohistochemistry, sections were collected on silanized slides (Dako-Cytomation, Glostrup, Denmark). After treatment at 60 °C for 24 h, sections were hydrated, pretreated with 98% formic acid for 1 min, followed by hydrating autoclaving for 30 min at 121 °C, and finally cooling overnight. Incubation with antibodies, plus avidin–biotin complex treatment and revelation, were carried out with Dako-Autostainer (Dako-Cytomation). Sections were treated with 6% normal goat serum (Vector, Burlingame, California, United States) in PBS for 30 min. Immunohistochemical detection of PrP was performed with mAb SAF84 (Spi-Bio, Montigny Le Bretonneux, France) at 2 μg/ml in PBS with 3% of normal goat serum (Vector) for 45 min. After washing with PBS, sections were treated with ABC Complex (Vector) for 45 min and with diaminobenzidine (Dako-Cytomation) for 7 min. Sections were counterstained with Mayer's haematoxylin. In each run, positive- and negative-control sections were included.
Sections for PET blot were collected on prewetted 0.45-μm-pore nitrocellulose membranes (Schleicher & Schuell, Dassel, Germany). Membranes were dried for 24 h at 55 °C. Membrane treatments, proteinase K digestion (50 μg/ml), and immunodetection were performed as described [50 (link)]. Monoclonal Ab SAF84 (1 μg/ml) was used as primary antibody.

Approximately 2 g of stool were collected from each study participant using a wide‐mouthed, leak‐proof, and clean stool cup. Because intestinal parasites are shed intermittently, study participants were requested to submit two stool samples for evaluation on 2 consecutive days. Stool samples were labeled with a unique code and transported to the Tefera Hailu Memorial Hospital Laboratory immediately after collection. For the direct saline method, an applicator stick was used to mix about 50 mg of feces with one or two drops of normal saline placed on a clean slide. A cover slip was used to create a thin, uniform suspension. The entire film was screened systematically for the presence of parasites. The remaining samples were then preserved using a modified acid‐fast technique and 10% formalin for formol‐ether concentration.
In addition, using an applicator stick, about 1 g of feces was placed in a clean 15‐mL conical centrifuge tube containing 7 mL of formalin‐saline for the formol‐ether concentration technique. The resulting suspension was filtered through a sieve into another conical centrifuge tube. The debris trapped in the sieve was discarded. After adding 3 mL of diethyl ether to the formalin solution, the contents were centrifuged at 3200 rpm for 3 min. The supernatant was discarded, and the tube was returned to its rack. A smear of the sediment was made on a clean glass slide and covered with a cover slip. Then, the entire area under the coverslip was systematically examined using ×10 and ×40 objective lenses. Furthermore, in the case of modified acid‐fast staining, the fecal smear is methanol‐fixed (1 min) and stained with carbol fuchsin for 5 min. Then it is rinsed with 50% ethanol followed by tap water. It is then decolorized with 1% sulfuric acid for 2 min, washed with tap water, and counter‐stained with alkaline methylene blue for 1 min. Finally, examine the oocyst of intestinal coccidian parasites under a light microscope using a 100‐fold magnification.