Recruitment of infective samples
Twenty -five patients attending Diagnostic and Research Unit of Parasitology (DRUP) at Kasr Al-Ainy school of medicine, the outpatient clinic of Abu El-Rish children hospital and the outpatient clinic of Theodor Bilharz Research Institute (TBRI) in the period from December 2019 to October 2020 were considered for the study. Two to three samples were collected from each patient. Patients with inflammatory bowel disease, gastrointestinal tumours were excluded as well as subjects who took antibiotics, proton pump inhibitors or non-steroidal anti-inflammatory drugs in the last 30 days. Subjects with persistent gastrointestinal manifestations more than 6 months were also excluded. All samples were recruited in sterile clean plastic cups taking care that the specimens were not contaminated with water or urine then they were subjected to the routine macroscopic & microscopic examination.
Isolation of the parasite and culture conditions
Positive stool samples containing Blastocystis were pooled together then emulsified in normal saline and strained through gauze into centrifuge tube. The tube was centrifuged at 2000 r.p.m. for 10 minutes, the supernatant was decanted. This process was repeated several times until the supernatant was totally clear.
The parasite was grown using Jones’ media supplemented with 10% horse serum and antibiotic solution (Penicillin G and Streptomycin in a concentration of 0.1%). The pH was accustomed to pH 7 with Na2HPO4, KH2PO4, and NaCl prior to autoclaving for sterilization at 121oC. For culture inoculation, a stool portion was transferred aseptically into the culture tubes with a clean glass rod and mixed with the culture medium. Each tube was labelled with the patient's name and number, and the date and incubated at 37ºC for 48 – 72 hrs. in the incubator.
Twenty laboratory bred male Swiss albino mice aged 3-4weeks age were supplied by the European Country Farms in Egypt and were affirmed in TBRI. Throughout the experiment, the mice were kept under standard experimental circumstances in a 12 h light/dark cycle and were kept on a standard diet containing 24% protein, 4% fat and about 4-5% fiber and water in the biological unit of TBRI under a temperature of 24°C. Periodic veterinary inspections were performed to approve that all animals were clear of common murine pathogens. Hygienic disposal was performed to remove mice waste and the dead animals.
Sample size was calculated by: Statistical analysis of ANOVA test as (according to G power analysis (v.9.3.1) α=0.05, sample size effect =0.4, 1-β=0.5, number of group 4, one way, total sample size =20, sample size =5 mice (Arifin and Zahiruddin, 2017). Group I (healthy control group) received neither infection nor lactose. Group II was infected by Blastocystis and received lactose diet. Group III were non-infected mice and received lactose diet (positive control). Group IV was infected with Blastocystis without lactose intake (negative control).
Induction of infection
Experimental mice were inoculated via intra oesophageal catheter with 104 cysts from Blastocystis culture (Moe et al., 1997) suspension in sterile saline. Weekly post-infection (p.i.), faecal samples from mice’s rectum were collected and subjected to parasitological examination by direct wet mount technique to detect Blastocystis infection.
Fourteen days p.i., Blastocystis was detected in stool with more than 8 cysts in the field indicating heavy infection of mice when lactose diet was initiated to group II and group III for 7 consecutive days (Silvia et al., 2002). Extrapolation of animal dose was calculated using the following formula (Nair and Jacob, 2016):
Twenty-one days post infection all mice were sacrificed by rapid decapitation of all groups by a well-trained laboratory technician. Mice were fastened overnight to minimize intestinal contents and parts of the small intestine were prepared for detection of lactase enzyme activity. They were stained with Haematoxylin and Eosin to detect any histopathologic or structural abnormalities in addition to immunostaining for detection of inflammatory (TNF-α) and apoptotic biomarkers (Bax and Bcl-2) in situ in collected tissue of the intestine.
Bioassay of lactase enzyme activity
Lactase activity was used as a biomarker of Blastocystis hominis induced mucosal damage. The activity of the enzyme was evaluated and defined as unites/mg of tissue protein.
Small intestinal tissue samples were accurately weighed; PBS (0.01 M, pH7~7.4) was added to the sample according to the ratio of weight (g): volume (mL) = 1:9; tissue samples were mechanically homogenized in ice water bath; all samples were then centrifuged at 3500 rpm for 10 min. Total protein concentration was investigated in the supernatant of homogenized tissue samples using NANODROP® 2000C spectrophotometer and the supernatant was stored at – 20 ºC.
The enzymatic reaction was carried out according to the lactase assay kit (Cat. no. E-BC-K131-S, Elabscience Biotechnology Inc, USA). Briefly, 5.5 mmol/L glucose standard solution was prepared together with the blank, samples and control. The substrate solution was added, mixed thoroughly and incubated at 37 ºC for 20 min. Afterwards, the stop solution was added and fully mixed, then all mixtures were centrifuged at 4000 r.p.m for 10 min. T he chromogenic agent was added to supernatant of each mixture, mixed well and incubated for 10 min. Each sample was measured in triplicate at 505 nm using spectrophotometer.
The amount of lactase in 1 mg of protein that hydrolyzes 1 nmol of lactose per minute at 37°C and pH 6.0 was defined as 1 unit according to the following formula:
The excised segments of the small intestine were cut both transversely and longitudinally, labelled and numbered then fixed in formalin 10%. Then the samples were impregnated in molten paraffin for 6 hours. After that samples were sectioned by microtome at 5 μm thickness (Drury and Wallington, 1980) and stained with Hematoxylin & Eosin stain (H&E).
Detection of inflammatory biomarker (TNF) and apoptotic biomarkers (Bax and Bcl-2) was performed in situ in the intestine. Tissue sections were deparaffinised, dehydrated in absolute ethanol for 15 seconds, and incubated sequentially in 2% (v/v) H202 in methanol for 30 to 45 seconds, 95% ethanol for 20 seconds, 70% ethanol for 20 seconds, distilled H20 for 1 minute, and PBS (120 mmol/L NaCI, 11.5 mmol/L NaH2PO4, 31.3 mmol/L KH2PO4, pH 7.4 to 7.6) for 5 minutes. Then the tissue sections were heated by microwaving in acidic buffer then washed twice in PBS for 5 minutes.
Tissue sections were pre-blocked for 30 to 45 minutes in Tris sodium potassium (TNK) solution (100 mmol/L Tris, pH 7.6 to 7.8; 550 mmol/L NaCI; 10 mmol/L KCI) containing 2% (w/v) bovine serum albumin, 0.1% Triton X-100, and 1% normal goat serum. We applied the following anti-rodent primary monoclonal antibodies: anti-Bax (1:1000 to 1:2000 v/v), anti-Bcl-2 (1:800 to 1:1500 v/v), and anti-TNF-α. Then the tissue sections were incubated overnight at room temperature. After washing with PBS, tissue sections were incubated for 1 hour with 2.8 pg/ml of biotinylated goat anti-rabbit antibody in the same buffer containing 0.5% normal mouse serum. Tissue sections were then washed and incubated for 30 to 45 minutes with an avidin-biotin complex reagent containing horseradish peroxidase (Vector) in TNK. Colour development was achieved by incubation for 10 minutes with a solution containing 3, 3'-diaminobenzidine and 0.1% (v/v) H202 in TNK buffer. Counterstaining was performed using hematoxylin. Slides were photographed with a light microscope equipped with a 35-mm camera with Ektar 100 film (Krajewskiet al., 1994). Median density, intensity, area percentage were measured and morphological analysis was carried using different magnification powers. Immunostained sections were evaluated in a blind fashion, with the help of a pathologist. Immunopositivity was scored from grade 1 to grade 4 by increasing the extent of immunostaining in 10 fields (1: 25%; 2: 25%–50%, 3: 50%–75%; 4: 75%–100%) and through the addition of the staining intensity grade: 1 (mild), 2 (moderate), and 3 (strong) immunoreactivity; the final grades were expressed as 1 (mild or weak), 2 (moderate), and 3 (strong).
Statistical analysis of data
The collected data were carefully revised, coded, tabulated and introduced to a personal computer using "Microsoft Office Excel Software" program (2010) for windows. The pre-coded data were then transferred to the Statistical Package of Social Science Software program, version 23 (IBM SPSS Statistics for Windows, Version 23.0. Armonk, NY: IBM Corp.) to be statistically analysed. Data were presented using range, mean, standard deviation (S.D.) for quantitative variables. Descriptive statistics were done in the form of the mean and the standard deviation (+/- S.D.) for the parametric numerical data. For the analytical statistics, paired t-test was used to assess the statistical significance among the experimental groups, P-value was considered significant at < 0.001. The results were represented in tables and graphs.