Study area and samples collection
A total of 200 soil samples were randomly collected, with 40 samples taken from each of the following locations: Feras market, garbage dumpster premises, Wadi-Gaza, public squares and gardens, and private house yards. The 90 water samples were collected from Wadi-Gaza stream, drinking water wells, and the city’s waste water treatment plant as shown in table (1), below which outlines the number of samples and the corresponding locations from which they were collected.
Table 1. Type, source, and number of samples collected in the current investigation.
Locality
|
Type of sample
|
Number of samples
|
Wadi – Gaza
|
Soil
Water (Running)
|
40
30
|
Trash dumpster premises
|
Soil
|
40
|
Houses yards
|
Soil
|
40
|
Public squares and gardens
|
Soil
|
40
|
Feras market
|
Soil
|
40
|
Sewage treatment plant
|
Water (Sewage)
|
30
|
Wells
|
Water (Household)
|
30
|
Total soil samples
|
|
200
|
Total water samples
|
|
90
|
Total environmental samples
|
|
290
|
Wadi Gaza is a water stream that originates in the mountains of Hebron (a Palestinian city located in the east of Gaza), flows through Gaza for about 8 kilometers, and then empties into the Mediterranean Sea (Madi 2005). Soil samples from Wadi Gaza were obtained precisely from the location where Wadi Gaza crosses the Salah El Deen bridge to roughly 1 km east, this is illustrated in Figure 1 below. The Feras Market, a significant local vegetable and other commodities market in downtown Gaza, was the second location where soil samples were collected. This market is regarded as the most popular and largest in the city. Furthermore, samples were obtained from soil near garbage bins in various active regions of the city. Following a rapid examination of certain residences where domestic and stray cats appeared to be in plenty, selected houses were picked for the purpose of collecting soil samples from their yards. Lastly, soil samples were collected from numerous public gardens and sites after monitoring them and taking into account their high cat population. Water samples were also obtained from various areas across the city. First, water samples were collected from Wadi-Gaza, which included a combination of sewage and rainfall. Water samples were also taken straight from a sewage treatment plant west of Gaza. Ultimately, they were collected from wells used for irrigation and household use, which are dispersed across the city and its neighboring suburbs.
Detection of T. gondii oocysts in soil and water:
From July through December of 2019, 290 soil and water samples were collected from the aforementioned locations. The methods used for analysis of soil samples were as described by Lass et al. (2009) and Colli et al. (2010). With minor modifications. Specifically, each soil sample was 300g in weight, taken at a depth of 2 to 5 cm below the surface layer, and dried for two days at room temperature. A total of 40 grams of each sample were mixed with 250 ml of distilled water before being filtered through a sieve with 75 μm pores. After straining, the liquid was placed into a 250 ml cylinder tube and left overnight. The next morning, 4 ml of liquid from the surface layer, (supernatant layer), was poured into 8 ml of sediment layer collected at the cylinder's base. This 12 ml mixture was then transferred to a centrifuge tube and centrifuged at 2500 rpm for 10 minutes. Following that, 2 ml of the mixture was collected from the tube's surface layer and mixed with the sediment in order to be microscopically examined using the Sheather's solution flotation technique (Lass et al. 2009; Colli et al. 2010). Regarding water samples, 90 samples containing 500 ml of water (30nfrom each of the locations mentioned previously) were collected. Samples were filtered using a Büchner funnel with a 0.45 μm nylon filter membrane. Using saline, the membrane was washed and then examined through the light microscope employing Sheather’s solution. Contaminated samples were put into Eppendorf tubes and stored at -20 °C for further PCR work. Meanwhile, samples showing no contamination were confirmed as negative using the PCR approach as reported by Papaiakovou et al (2019). (Papaiakovou et al. 2019)
Molecular Identification and Genotyping of T. gondii
DNA Extraction
The Wizard Genomic DNA extraction kit A1120 (Promega, Madisson, Wisconsin, USA) was used as directed by the manufacturer to extract DNA from the resuspended pellets. To easily detect the specified gene from the purified DNA, the final DNA pellets were resuspended in a 30 μL TE buffer (10 mMTris, 1 mM EDTA, pH 7.2) and preserved at -20 °C until the PCR procedure was completed (Amairia et al. 2016).
PCR Primers and Conditions
The PCR reaction was performed using a set of primers-TOXO1 (5' GGA ACT GCA TCC GTT CAT GAG 3') and Toxo2 (5' TCT TTA AAG GGT TGG TGG TC 3') reported by Lass et al. (2009). These premiers were particularly designed to target the 194 bp region of the 35-fold-repetitive B1 gene. To validate positive results, all positive samples were reexamined using TOXO-F (5' AGG GGA GGG TGA GGA TGA 3') and TOXO-R (5' TGG TCT CGT CTG GAT CGC AT 3') primers. These are especially unique to a 200 to 300-fold-repetitive element (REP) sequence segment (AF146527). Following that, all components were placed in the AccuPower PCR PreMix tube (Bioneer Corporation-Hylabs). For each isolate, PCR amplification of TOXO genes was carried out in a thermal cycler (Biometra, Germany). The following conditions were considered: a 5-minute initial denaturation phase at 95 °C, thermocycling for 30 cycles, with each cycle consisting of 30 seconds at 94 °C followed by 30 seconds at 55 °C for annealing, 45 seconds at 72 °C for extension, and a final extension cycle of 10 minutes at 72 °C (Lass et al. 2009; Hassanain et al. 2013; Dardona et al. 2021). Distilled water was used as a negative control, while a previously identified and well-defined T. gondii sample was used as a positive control. The PCR work was conducted in the molecular biology laboratory of the health sciences department at the Islamic University of Gaza.
Genotyping
The PCR-RFLP method described by Norouzi et al. (2016) was implemented for genotyping four of the PCR-positive samples. Briefly, a 791 bp DNA fragment was amplified using the GRA6 primers (5`-GTAGCGTGCTTGTTGGCGAC-3΄) and (5`TACAAGACATAGAGTGCCCC-3΄). The fragment was then digested by the restriction enzyme MseI. The digestion products distinguish the different types of Toxoplasma gondii. Products of 168 and 544 bp, 75 and 623 bp, and 97 and 544 bp fragments identify type I, type II and type III, respectively (Norouzi et al. 2016).
Statistical Analysis
Analysis performed on the results drawn from the subject study was carried out through using the SPSS (USA, II, Chicago, SPSS Inc) software package v. 15.0. Chi-square tests were performed to highlight the comparison between contamination of T. gondii oocysts and two other variables, the environmental samples from which they were obtained, and the months in which they were collected (July to December of 2019).A p-value less than 0.05 was considered significant.