Toxoplasma gondii detection and genotyping in the Environmental matrices: soil and water in Gaza, Palestine

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For this purpose290 environmental samples, including 200 from soil and 90 from water, were collected. Soil samples were collected from Wadi-Gaza, vicinity of trash dumpsters, residential home yards, open-air marketplaces, public squares and gardens. Water samples were collected from Wadi-Gaza, wells used for drinking and irrigation. The samples were examined utilizing PCR and Sheather's solution techniques and genotyping of 4 samples was performed by PCR-RFLP. The results revealed that out of the 290 investigated samples, 31 (10.68%) proved contaminated by Sheather’s solution, whereas 22 (7.58%) were positive by PCR. Genotyping results showed that all of the four tested samples belonged to T. gondii Type I. The highest rate of contamination was detected in the samples collected from residential house yards, while public squares and gardens represented the lowest rate. Regarding the water samples, T. gondii oocysts were detected only in the samples collected from Wadi-Gaza. This work confirms the presence of Type I T. gondii in the soil and water of the study area, with soil contamination being heavier than that of water. Toxoplasma gondii oocysts Genotype soil water Gaza-Palestine Figures Figure 1 Figure 2 Figure 3 Introduction T. gondii is an obligate intracellular protozoan replicating with a three-stage life cycle that is infectious to all warm-blooded animals(Robert-Gangneux and Dardé 2012 ; Maenz et al. 2014 ).Sexual reproduction occurs exclusively in the feline definitive host, leading to the formation of oocysts that are excreted in their feces. After sporulation in the environment, oocysts (the environmental stage) become infective. T. gondii oocysts are resistant to extreme environmental, physical, and chemical conditions and can survive for a long time in the environment (Robert-Gangneux and Dardé 2012 ; Dumètre and Dardé 2003 ). Ingestion of sporulated oocysts by intermediate hosts results in the release of sporozoites, which pass through the intestinal epithelium and develop into tachyzoites. This is followed by tachyzoite spread through body fluids, including enormous and rapid asexual reproduction in nucleated cells of the host (Robert-Gangneux and Dardé 2012 ; Maenz et al. 2014 ). This stage of infection is distinguished by a significant inflammatory response and host tissue damage, which can lead to clinical signs of toxoplasmosis. After the host's immunological response has progressed, replication slows, and the now-called bradyzoites settle in host cells of the retina, brain, skeletal, and cardiac muscles (Blanchard et al. 2015 ). The parasite may avoid the immune system and lives a lifespan within the host by forming tissue cysts. Humans are susceptible to T. gondii infection, which may be acquired horizontally by ingestion of oocysts via infected food (vegetables, fruits, and water), infection after gardening, or direct contact with cat feces. Consumption of raw or undercooked meat harboring highly infectious tissue cysts, can also result in oral-alimentary transmission)Tenter et al. 2000 ; Dias et al. 2005 ). A Research conducted in 1999 showed that drinking water functioned as a vehicle for oocyst transmission among Jains, a vegetarian group in India (Hall et al. 1999 ). Besides, a large outbreak of toxoplasmosis was recorded in Brazil in 2001 and 2002 as a result of consuming water from municipal sources (De Moura et al. 2006 ). Certainly, a variety of environmental conditions contribute to the spread of T. gondii . These serve as a vector for the parasite's transmission to surface water sources, some of which include heavy rains and winds(Mac Kenzie et al. 1994 ). Toxoplasmosis is the third most common food-borne disease, behind salmonellosis and listeriosis (Jones et al. 2001 ; Dubey and Jones 2008 ), as well as being one of the most widespread zoonotic diseases. It is believed that about 30% of the human population is infected with T. gondii (Maenz et al. 2014 ). While the seroprevalence of this parasite is expected to be greater than 50% in regions where raw or undercooked meat is often consumed. Furthermore, high seroprevalence is prevalent in tropical parts of Latin America and Africa, where cat populations are considerable and climatic conditions are favorable for oocyst survival (Jones et al. 2001b ; Di Carlo et al. 2008 ; Dubey et al. 2009 ). It is estimated that 15% of childbearing women aged 25 to 44 years in the United States are infected with T. gondii . Meanwhile, it is reported that between 400 and 4,000 cases of congenital toxoplasmosis occur annually (Jones et al. 2001b ). T. gondii was also ranked fourth in an assessment of the worldwide significance of 24 food-borne parasites based on socioeconomic and trade impacts(FAO/WHO 2014 ). Furthermore, occasional outbreaks of waterborne toxoplasmosis have been observed in various places of the world e.g., Panama, British Columbia, and Brazil (Benenson et al. 1982 ; Bowie et al. 1997 ; Lass et al. 2009 ). Despite the fact that contaminated drinks and foods constitute the significant risk factors, a variety of other factors, including poor socioeconomic and educational levels, do also contribute to the occurrence of toxoplasma outbreaks (Ferreira et al. 2018 ). Numerous studies have been conducted to investigate the seroprevalence of T. gondii infection in pregnant and abortion-experiencing women in Palestine. According to Al-Hindi and Lubbad (2009), the occurrence of T. gondii specific antibodies was 12.8% for IgM among 312 women who had abortions in Gaza-Palestine (Al-Hindi and Lubbad 2009).While among 204 pregnant women in Hebron-Palestine, a high seroprevalence of 27.9% was recorded with women from rural areas being more affected than those from urban areas (Nijem and Al-Amleh 2009 ). Furthermore, in Gaza, Al-Jarousha ( 2012 ), reported 30.9% seropositivity among 255 pregnant women (Al-Jarousha 2012 ). The subject study's goal was to detect T. gondii oocysts in environmental samples, such as soil and water, in Gaza, Palestine, using conventional (Sheather's solution) and molecular (PCR) techniques, as well as to determine the genotype of some of the isolates. Materials And Methods 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. Results The present study results revealed that T. gondii oocysts had a conspicuous prevalence in some of the examined environmental samples. More precisely, 22 (7.58%) samples out of the 290 tested water and soil samples were found to be contaminated with oocysts according to the PCR technique, while 31 (10.68%) were positive according to Sheather’s solution. Of the PCR-positive samples 18 were soil samples and four water samples. In the case of Sheather's solution, 26 of the 31 contaminated samples were from soil and 5 were from water samples. The distribution and comparison of the data obtained from the two techniques employed in the study are shown in Table (2) below. The current investigation concluded that water samples collected from Wadi Gaza were the only source of oocyst contaminated water, despite the fact that PCR and Sheather's solution revealed 4 and 5 contaminated samples, respectively. In addition, T. gondii oocysts were not detected in samples collected from wells or the sewage treatment facility. On the other hand, contamination has been documented by every source of soil samples. The greatest rate of contamination was seen in samples acquired from residential home yards, with 9 and 7 samples detected by Sheather's and PCR, respectively. Feras market and Wadi Gaza ranked second and third in terms of the level of oocyst contamination, with 5 samples per Sheather's solution for both of them, and 5 and 3 samples per PCR, respectively. Following that, soil samples were collected from the premises of garbage dumpsters, with 4 and 2 samples, respectively, according to Sheather's and PCR. Lastly, samples collected from public gardens and squares had the lowest contamination rate, with two samples per Sheather's solution and one sample per PCR (Table 2 ). As shown in Table (2), there is a significant difference in the incidence of T. gondii oocysts in the various environmental samples studied by both techniques and their associated location sources. In terms of the month in which the samples were gathered, the subject study's findings showed an interesting trend. The highest number of contaminated samples was found in December, followed by November, October, September, August, and July (Table 3 ). The month of July had the lowest rate of contaminated samples. Table 2 Prevalence of T. gondii oocysts in the environmental samples, based on the source of sample using both PCR and Sheather’s techniques. Environmental Sample (Type) Sample (No) Positive (Sheather’s) Positive (PCR) Wadi –Gaza (Running water) 30 5 4 Sewage treatment plant (sewage water) 30 0 0 Wells (drinking and irrigation) 30 0 0 Total water samples 90 5 (5.55%) 4 (4.44%) Wadi – Gaza (Soil) 40 5 3 Trash dumpster premises(Soil) 40 4 2 House yards (Soil) 40 9 7 Public gardens (Soil) 40 3 1 Feras market (Soil) 40 5 5 Total soil samples 200 26 (13.00%) 18 (9.00%) Total (Water and Soil) 290 31 22 % positive 10.68% 7.58% χ2 (PCR) = 16.79 P = 0.01 P < 0.05. Statistically significant χ2 (Sheather’s ) = 14.86 P = 00.038 P < 0.05. Statistically significant The relationship between sample contamination with oocysts by both techniques and the months in which samples were collected had demonstrated no statistical significance, as indicated by the findings in table (3) below. Figures (2) below presents a photograph of ethidium bromide stained agarose gel illustrating some of the PCR-positive T. gondii oocyst samples. Table 3 Monthly prevalence of Toxoplasma gondii oocyst in the environmental samples in Gaza city during the period July-December 2019. Month Samples (No) Soil (No) Water (No) (+ Ve) Sheather’s (+ Ve) PCR July 43 28 15 3 1 August 67 41 26 2 2 September 20 12 8 4 2 October 57 50 7 7 4 November 60 42 18 8 6 December 43 27 16 7 7 Total 290 200 90 31 (10.68%) 22 (7.58%) χ2 ( PCR) = 9.047 P = 00.107 P > 0.05. Not Statistically significant χ2 (Sheather’s ) = 8.600 P = 00.126 P > 0.05. Not Statistically significant Regarding the PCR-RFLP genotyping, figure (3) below shows that the MseI digestion products are 544 and 168 bp thus indicating that the four samples tested are of Toxo-type I. Discussion The study's main purpose was to detect T. gondii oocysts in environmental samples, particularly soil and water. Given that the majority of T. gondii infections in pregnant women occur as a result of soil contact, as soil is assumed to be an important environmental factor in human infections (Ajmal et al. 2013 ). According to a European research, 17% of T. gondii infections are caused by contact with contaminated soil, as demonstrated by a direct survey of women infected with the parasite. Because oocysts can remain infectious in soil for more than a year, contaminated soil is seen as a serious risk factor in the parasite's spread (Lass et al. 2009 ). Indeed, soil is an important environmental source of T. gondii infection in humans and animals (Cook et al. 2000 ; dos Santos et al. 2010 ). This is corroborated by the findings of the present investigation, which revealed the existence of oocysts in all of the locations where soil samples were taken. Because oocysts are exclusively excreted through cat feces, and every site where samples were taken included cats. Because stray cats are found in almost every area of Gaza, particularly in Wadi Gaza and the surrounding agricultural lands, where there are abundant food sources and this explains the highest rate of contamination detected in Wadi-Gaza and residential home yards. Many of the cats found in those areas tend to be infected with T. gondii e.g. , Al-Hindi et al ( 2019 ). documented the occurrence of T. gondii oocysts in the feces of some stray cats in a study conducted in Khanyounis Governorate, located in the south of Gaza City (Al-Hindi et al. 2019 ). Since they are stray cats, they may easily move from one region to another, making them significant risk factors for T. gondii oocyst transmission and contamination of soil and flowing water. This is understandable considering that cats, as previously stated, play a role in the dissemination of oocyst. The results of the current study are consistent with those of a comparable study conducted in Poland, which showed the existence of oocysts in 18 (17.8%) of 101 soil samples using the PCR technique. Furthermore, another study conducted in China reported that 34 (12.69%) of 286 soil samples were contaminated with T. gondii oocysts (Lass et al. 2009 ; Du et al. 2012 ). The present study's findings revealed that the surface water stream, Wadi Gaza, was contaminated with oocyst. This water stream is a mix of rain and sewage water that flows from east to west across Gaza and into the Mediterranean Sea. The presence of cats beside the water stream can be blamed for the contamination of this water. This creek provides nutritional resources to cats due to people's terrible habit of dumping waste along the stream, as well as the existence of local and immigrant birds, rats, and decaying animal corpses. Besides, other environmental elements that impact the spread of oocysts in surface water sources include rain, wind, soil, and mechanical vectors such as insects, flies, and earthworms (Dumètre and Dardé 2003 ). Other water samples collected from other locations were found to be free of oocyst contamination. This can be attributed to the absence of infected cats and other variables previously identified as being important for oocyst dissemination. The wells and the water treatment plant were correctly closed to avoid the existence of cats or the effect of other factors capable of spreading these oocysts. Another study conducted in the study area regarding the detection of parasitic contamination in the water used for kitchen purposes and carried out by AL-Hindi et al. (2021) confirms the results of the current study, as their study did not record the occurrence of T. gondii oocysts in this water and only recorded the occurrence of cryptosporidium (AL-Hindi et al. 2021). It is also commonly known that the water utilized for household purposes in that area is sourced from wells distributed around the area. On the other hand, T. gondii was not regarded as a serious aquatic pathogen, in contrast to cryptosporidium (Mac Kenzie et al. 1995 ). In view of current toxoplasmosis outbreaks in people, the topic study emphasized the significance of oocyst contamination in environmental samples. The findings of the current study were comparable to those of a study conducted in France, where oocysts were found in 37 (7.7%) of 482 environmental water samples (Aubert and Villena 2009 ). According to the interpretations formed by the author of the French study based on the study's data, the major cause of this contaminated water was contaminated soil rinsed by rain water. This view, in fact, confirms the conclusion obtained from the subject study's findings. Rainwater combined with dirt and sewage may have played a significant part in the oocyst contamination of Wadi Gaza, the primary surface water stream that runs for a long distance before emptying into the Mediterranean. To investigate the authenticity of this phenomenon, water samples from Wadi Gaza were obtained just one kilometer before its endpoint, the Mediterranean Sea. Given the length of the stream, this collection point enabled the subject researchers to recognize and quickly investigate the potential impact of soil and other environmental elements on water contamination. Not to mention, the topic investigation indicated a congruent conclusion based on comparable results obtained from a study conducted in Brazil, which discovered 3 (7.7%) of 39 environmental water samples collected from surface water streams were utilized largely for drinking water (Galvani et al. 2019). This research, as well as the previous ones, corroborates the existence of oocyst contamination in surface water sources, which is regarded as a high contamination rate, particularly when utilized for irrigation. The current investigation is one of the few that have been conducted to detect oocyst contamination or even parasites in the Gaza environment. In their study, Dardona et al. ( 2021 ) recorded the occurrence of T. gondii oocysts in raw leafy vegetables, which are commonly consumed raw in Gaza. One of their interpretations of their findings is that soil or water contamination, as well as some environmental factors, play a significant role in oocyst spread to vegetables (Dardona et al. 2021 ). Another study conducted in the study region by Hilles et al. ( 2013 ), recorded the contamination of the Gaza shoreline with certain parasites without mentioning T. gondii among the detected parasites (Hilles et al. 2013 ). Nonetheless, the small differences in the results obtained by different research conducted in different cities and countries might be attributable to a variety of factors. First, there are the various detection procedures used; second, there are the various environmental elements that aid in the transmission of oocysts; and finally, there is a variation in cat population rates and presence in the neighborhood of where the samples were gathered. Even before 1970, when the T. gondii life cycle was established, there was a disagreement over the relevance of the epidemiological components of T. gondii infection, whether by ingestion of oocysts or tissue cysts (Jackson and Hutchison 1989 ). Although the relationship between the contamination of soil and water samples and the month in which the samples were collected was not found to be statistically significant, it can be noted that the highest contamination rates were during the months of November and December, and the lowest levels of contamination were during the months of July and August. This is easily explained by the fact that the quantity of rain that falls during the winter raises the average water level in the valley's stream, increasing the speed of the valley's run-off, which transports possible sources of contamination. Furthermore, the high level of the stream causes flooding, and the water floods the agricultural lands located on the valley's boundaries. This further increases the rate of contamination. Furthermore, the possibility of the previously stated environmental factors, such as rain water and wind, in the transmission and dissemination of Toxoplasma oocysts rises during the winter (Dumètre and Dardé. 2003). The first observation made upon discovery was that both vegetarians and non-vegetarians are susceptible to T. gondii infections, suggesting that carnivorism was not the only source of T. gondii infections (Dubey 2009 ; Shapiro et al. 2019 ). The degree of T. gondii infection in cats is determined in conjunction with the degree of infection in birds, rodents, and other sources of cat food. Just as the incidence of oocysts in the environment is increasing, contamination of surface water leading to sea water constitutes a concerning issue (Sibley and Boothroyd 1992 ; Afonso et al. 2007 ). Infected cats defecate millions of oocysts in soil after swallowing only one tissue cyst or bradyzoite, and the sporulated oocysts stay infectious in soil for up to 18 months, independent of temperature exposure. Despite this, finding oocysts with a light microscope was sufficient for significantly contaminated samples. However, the molecular technique is more tempting because of its high sensitivity and rapidity in detecting T. gondii by PCR amplification of the B1 gene (Dumètre and Dardé, 2003 ; Dubey, 2009 ).The current study, on the other hand, employed a PCR-RFLP assay on the GRA6 gene to genotype four DNA isolates. It is worth noting that this is the first study in the research area that tackled the genotyping of T. gondii isolates, and GRA6 was used in the parasite genotyping process because it is a coding region with a larger and more diverse polymorphism than other regions such as SAG1, SAG2, and GRA4 (Norouzi et al. 2016 ). Furthermore, the current study's findings revealed that all of the analyzed oocyst DNA isolates belonged to the Toxo-Type I. The genotyping of the parasite is regarded as a critical component in generating the disease, as demonstrated by several mouse model trials, and it has been documented in greater detail that genotype I strains are very virulent, but the other kinds are not harmful to a considerable extent (Sibley and Boothroyd 1992 .). It is crucial to note that T. gondii DNA isolates were categorized based on their virulence in outbred mice, but the first phylogenetic investigations of T. gondii strains revealed that their complexity was significantly lower than predicted (Dardé et al. 1992 ; Sibley et al. 1992 ). One of the most essential aspects of genotyping is its application in tracking epidemics (Ajzenberg et al. 2004 ). It has been demonstrated that there are large geographical variations in the distribution of T. gondii genotypes. For example, certain investigations performed in Spain and Portugal have reported the existence of types I and III in these locations (Fuentes et al. 2001 ; de Sousa et al. 2006 ). On the other hand, the findings of investigations conducted to assess the genotyping of T. gondii for isolates in both Crete and Cyprus reported the existence of type III in both areas (Messaritakis et al. 2008 ). To round out the findings, French research revealed that only four of 86 T. gondii isolates belonged to genotype I. On the contrary, genotype I was shown to be extremely dominant in one of the investigations done on the isolated strains of CSF of eight patients with acquired immunodeficiency in the United States, as the findings of this study revealed that the majority of them were infected with T. gondii genotype I (Ajzenberg et al. 2004 ; Khan et al. 2005 ). On the other hand, in a study conducted in Brazil to identify the genotyping of T. gondii isolates reported in water supply samples, after genotyping them based on the SAG2 locus, it was reported that genotype I is very pathogenic (De Moura et al. 2006 ). Conclusion The current study concluded that T. gondii oocyst contamination in soil is greater than in water. The highest rate of soil contamination was detected in samples obtained from private home yards, whereas the lowest rate was reported in public squares and gardens. The contaminated water samples, on the other hand, are solely from flowing water samples gathered from Wadi Gaza, whereas water collected from other sources was confirmed to be clear of contamination. Finally, this investigation revealed that all genotyped samples belonged to T. gondii type I. Declarations Acknowledgment The authors would like to express their thanks and gratitude to Mr. Muhammad Ashour for his technical assistance in the PCR work. Competing interests Not applicable Funding Not applicable Availability of data and material The authors affirm that the data supporting this study are available within the article and that raw data supporting the study's findings are available from the first author, Zuhair Dardona, upon reasonable request. Authors' contributions 1- Zuhair Dardona ( Practical work– writing – statically analysis). 2-Adnan Al-Hindi ( writing – review) 3- Samia boussaa ( writing- review) 4- Mohamed Hafidi (Review) 5- Ali boumezzough (Reviw) 6- Fadel Sharif ( Genotyping – review) Ethics approval On August 5, 2019, the ethical research committee of the Islamic University of Gaza approved an ethical clearance for this study, which planned to gather water and soil samples from the study area. Consent to participate All of the authors have willingly agreed to take part in this research project. 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Dardé ML, Bouteille B, Pestre-Alexandre M (1992) Isoenzyme analysis of 35 Toxoplasma gondii isolates and the biological and epidemiological implications. J Parasitol 78:786–94. Dardona Z, Al Hindi A, Hafidi M, Boumezzough A, Boussaa S (2021) Occurrence of Toxoplasma gondii on Raw Leafy Vegetables in Gaza, Palestine. J Food Prot 842:255–61. De Moura L, Bahia-Oliveira LM, Wada MY, Jones JL, Tuboi SH, Carmo EH, Ramalho WM, Camargo NJ, Trevisan R, Graça RM, Da Silva AJ (2006) Waterborne toxoplasmosis, Brazil, from field to gene. Emerging Infect Dis 122:32–36. de Sousa S, Ajzenberg D, Canada N, Freire L, da Costa JC, Dardé ML, Thulliez P, Dubey JP (2006) Biologic and molecular characterization of Toxoplasma gondii isolates from pigs from Portugal. Vet parasitol 1352:133–136. Di Carlo P, Romano A, Schimmenti MG, Mazzola A, Titone L (2008) Materno-fetal Toxoplasma gondii infection: critical review of available diagnostic methods. Infez Med 161:28–32. Dias RA, Navarro IT, Ruffolo BB, Bugni FM, Castro MV, Freire RL (2005) Toxoplasma gondii in fresh pork sausage and seroprevalence in butchers from factories in Londrina, Paraná State. Brazil Rev Inst Med Trop Sao Paulo 474:185–189. dos Santos TR, Nunes CM, Luvizotto MC, de Moura AB, Lopes WD, da Costa AJ, Bresciani KD (2010) Detection of Toxoplasma gondii oocysts in environmental samples from public schools. Vet parasitol 1711-2:53–57. Du F, Feng HL, Nie H, Tu P, Zhang QL, Hu M, Zhou YQ, Zhao JL (2012) Survey on the contamination of Toxoplasma gondii oocysts in the soil of public parks of Wuhan, China. Vet parasitol 1842-4:141–146. Dubey JP, Jones JL (2008) Toxoplasma gondii infection in humans and animals in the United States. Int J Parasitol 3811:1257–12578. Dubey JP, Lindsay DS, Lappin MR (2009) Toxoplasmosis and other intestinal coccidial infections in cats and dogs. Vet Clin North Am Small Anim 396:1009–10034. Dubey JP (2009) History of the discovery of the life cycle of Toxoplasma gondii . Int J Parasitol 398: 877–882. Dumètre A, Dardé ML (2003). How to detect Toxoplasma gondii oocysts in environmental samples? FEMS Microbiol Rev 275:651–661. El-Hallaq MA (2019) Studying the Impact of Pollution from Wadi Gaza on the Mediterranean Sea Using GIS and Remote Sensing Techniques. Int J Remote Sens 81: 40–50. FAO/WHO (2014) Multicriteria-based ranking for risk management of food-borne parasites: report of a Joint FAO/WHO expert meeting, 3–7 September 2012, FAO Headquarters, Rome, Italy. FAO, World Health Organization. https://apps.who.int/iris/handle/10665/112672 . Ferreira FP, Caldart ET, Freire RL, Mitsuka-Breganó R, Freitas FM, Miura AC, Mareze M, Martins FD, Urbano MR, Seifert AL, Navarro IT (2018) The effect of water source and soil supplementation on parasite contamination in organic vegetable gardens. Rev Bras DE Parasitol Vet 3027:327–37. Fuentes I, Rubio JM, Ramírez C, Alvar J (2001) Genotypic characterization of Toxoplasma gondii strains associated with human toxoplasmosis in Spain: direct analysis from clinical samples. J Clin Microbiol 394:1566–1570. Galvani AT, Christ AP, Padula JA, Barbosa MR, de Araújo RS, Sato MI, Razzolini MT. 2019 Real-time PCR detection of Toxoplasma gondii in surface water samples in São Paulo, Brazil. Parasitol Res 1182:631–640. Hall SM, Pandit A, Golwilkar A, Williams TS (1999) How do Jains get Toxoplasma infection? Lancet 3549177:486–487. Hassanain MA, El-FadalyHA, Hassanain NA, Shaapan RM, Barakat AM, Abd El-Razik KA (2013) Serological and molecular diagnosis of toxoplasmosis in human and animals. World J Medical Sci 94:243–247. Hilles AH, Al-Hindi AI, AbuSafieh YA (2013) Is Gaza sandy shoreline region contaminated with human gastrointestinal parasites? Jordan J Biol Sci 6(3): 205–210. Jackson MH, Hutchison WM (1989) The prevalence and source of Toxoplasma infection in the environment. Adv Parasitol 28: 55–105. Jones JL, Kruszon-Moran D, Wilson M, McQuillan G, Navin T, McAuley JB (2001b) Toxoplasma gondii infection in the United States: seroprevalence and risk factors. Am J Epidemiol 1544:357–365. Jones JL, Lopez A, Wilson M, Schulkin J, Gibbs R (2001) Congenital toxoplasmosis: a review. Obstet Gynecol Surv 565: 296–305 Khan A, Su C, German M, Storch GA, Clifford DB, Sibley LD (2005) Genotyping of Toxoplasma gondii strains from immunocompromised patients reveals high prevalence of type I strains. J Clin Microbiol 4312: 5881–5887. Lass A, Pietkiewicz H, Modzelewska E, Dumètre A, Szostakowska B, Myjak P (2009) Detection of Toxoplasma gondii oocysts in environmental soil samples using molecular methods. Eur J Clin Microbiol Infect Dis 286:599–605. Mac Kenzie WR, Hoxie NJ, Proctor ME, Gradus MS, Blair KA, Peterson DE, Kazmierczak JJ, Addiss DG, Fox KR, Rose JB, Davis JP (1994) A massive outbreak in Milwaukee of Cryptosporidium infection transmitted through the public water supply. N Engl J Med 3313: 161–167. Mac Kenzie WR, Schell WL, Blair KA, Addiss DG, Peterson DE, Hoxie NJ, Kazmierczak JJ, Davis JP (1995) Massive outbreak of waterborne Cryptosporidium infection in Milwaukee, Wisconsin: recurrence of illness and risk of secondary transmission. Clin Infect Dis 211:57–62. Madi MI (2005) Algae and wild plants of Gaza Strip. Manara Bookshop and Press, Gaza Strip, Palestine. Maenz M, Schlüter D, Liesenfeld O, Schares G, Gross U, Pleyer U (2014) Ocular toxoplasmosis past, present and new aspects of an old disease. Prog Retin Eye Res 39:77–106. Messaritakis I, Detsika M, Koliou M, Sifakis S, Antoniou M (2008) Prevalent genotypes of Toxoplasma gondii in pregnant women and patients from Crete and Cyprus. Am J Trop Med 792: 205–209. Nijem KI, Al-Amleh S (2009) Seroprevalence and associated risk factors of toxoplasmosis in pregnant women in Hebron district, Palestine. East Mediterr Health J 155:1278–1284. Norouzi M, Tabaei SJ, Niyyati M, Saber V, Behniafar H (2016) Genotyping of Toxoplasma gondii strains isolated from patients with ocular toxoplasmosis in Iran. Iran J Parasitol 113:316–324. Papaiakovou M, Wright J, Pilotte N, Chooneea D, Schär F, Truscott JE, Dunn JC, Gardiner I, Walson JL, Williams SA, Littlewood DT (2019) Pooling as a strategy for the timely diagnosis of soil-transmitted helminths in stool: value and reproducibility. Parasites Vectors 121:1–3. Robert-Gangneux F, Dardé ML (2012) Epidemiology of and diagnostic strategies for toxoplasmosis. Clin Microbiol Rev 252:264–96. Shapiro K, Bahia-Oliveira L, Dixon B, Dumètre A, de Wit LA, VanWormer E, Villena I. (2019) Environmental transmission of Toxoplasma gondii : Oocysts in water, soil and food. Food Waterborne Parasitol 15: e00049. Sibley LD, Boothroyd JC (1992) Virulent strains of Toxoplasma gondii comprise a single clonal lineage. Nature 3596390: 82–85. Sibley LD, LeBlanc AJ, Pfefferkorn ER, Boothroyd JC (1992) Generation of a restriction fragment length polymorphism linkage map for Toxoplasma gondii . Genetics 1324:1003–1015. Tenter AM, Heckeroth AR, Weiss LM (2000) Toxoplasma gondii : from animals to humans. Int J Parasitol 3012-13:1217–1258. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-1538884","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":98288821,"identity":"80c0eb2f-d19c-44b5-85f7-6b6c690aac02","order_by":0,"name":"Zuhair Dardona","email":"","orcid":"","institution":"Cadi Ayyad University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Zuhair","middleName":"","lastName":"Dardona","suffix":""},{"id":98288823,"identity":"9c884dac-470d-4434-995e-c489b0052891","order_by":1,"name":"Adnan Al-Hindi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4UlEQVRIiWNgGAWjYDACCcYGBh4DGx4YH8glTksaSVqAmIfhMJxPWAv/7Oa2B28KzssYnF/8+MMPBhvZDQd4D3/Aa8mdg+2Gcwxu8xjceGZg2MOQZrzhAF+aBF5rbiS2SfOAtRwwSAC6MHHDAR4zvDrkIVrOAbUc/3DwD8N/kBZjvA4zgGg5wGNwvsewmYfhAEiLAV6HGQK1SM4xSOaRvMFTzCxjkGw88zABv8jdSH8m8eaPnT3f+eObP76psJPtO96LP8QQQCIB5E4gZuYhoBIO+A/AWERrGQWjYBSMghECAIMnTaGLGfNkAAAAAElFTkSuQmCC","orcid":"","institution":"Islamic University of Gaza","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Adnan","middleName":"","lastName":"Al-Hindi","suffix":""},{"id":98288826,"identity":"82f61f9c-2ec0-41ee-b971-ff2c41e450e9","order_by":2,"name":"Mohamed Hafidi","email":"","orcid":"","institution":"Cadi Ayyad University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Mohamed","middleName":"","lastName":"Hafidi","suffix":""},{"id":98288829,"identity":"d08b49c1-44f1-499f-9319-953b66315294","order_by":3,"name":"Ali Boumezzough","email":"","orcid":"","institution":"Cadi Ayyad University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ali","middleName":"","lastName":"Boumezzough","suffix":""},{"id":98288831,"identity":"2b3e364d-8ec5-4590-b58d-acc45aaa6013","order_by":4,"name":"Fadel Sharif","email":"","orcid":"","institution":"Islamic University of Gaza","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Fadel","middleName":"","lastName":"Sharif","suffix":""},{"id":98288832,"identity":"04c40d39-ff34-4ac5-b784-93c0b1b51210","order_by":5,"name":"Samia Boussaa","email":"","orcid":"","institution":"Islamic University of Gaza","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Samia","middleName":"","lastName":"Boussaa","suffix":""}],"badges":[],"createdAt":"2022-04-08 23:44:05","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-1538884/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-1538884/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":20398743,"identity":"c4e99179-1625-4bcb-8ee5-a6cd5d40f171","added_by":"auto","created_at":"2022-04-15 19:58:02","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":83444,"visible":true,"origin":"","legend":"\u003cp\u003eThe location of Wadi Gaza (El-Hallaq\u0026nbsp;2019), the start of the arrow indicates the site of sample collection, whereas the arrow head points to the sea.\u003c/p\u003e","description":"","filename":"Fig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1538884/v1/ec3063e01eff5821704da33e.jpg"},{"id":20398741,"identity":"4a13ee4a-cb53-4ae1-86d6-a031e337270b","added_by":"auto","created_at":"2022-04-15 19:58:02","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":40109,"visible":true,"origin":"","legend":"\u003cp\u003eThe recovery of \u003cem\u003eT. gondii\u003c/em\u003e oocysts from experimentally contaminated water and soil samples. The B1 amplification products were placed on a 2% gel agarose. Lane 1, molecular weight size marker (50 bp, Bioline, Italy). Lane 2 is the negative control. Lane three is a positive control. Negative control (lane 4) (no template control). Lanes 5-8 positive samples contaminated with \u003cem\u003eT. gondii\u003c/em\u003e. Lane 9 indicates a negative sample\u003c/p\u003e","description":"","filename":"Fig2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1538884/v1/fdfd6ea3eef80ad2cc5c3d93.jpg"},{"id":20398742,"identity":"2ed4971b-07c3-4848-b34a-316587823bb6","added_by":"auto","created_at":"2022-04-15 19:58:02","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":47198,"visible":true,"origin":"","legend":"\u003cp\u003eA photograph of ethidium bromide stained agarose gel showing the PCR-RFLP pattern of the GRA6 amplified fragment after \u003cem\u003eMseI\u003c/em\u003e endonuclease digestion (168 and 544 bp bands). This pattern corresponds to type I \u003cem\u003eT. gondii\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"Fig3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1538884/v1/0fa137aee8c759debf8cfec1.jpg"},{"id":21210620,"identity":"ce33d5fb-fc04-4a9b-961c-a4af71a8519f","added_by":"auto","created_at":"2022-05-09 03:44:13","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":538745,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1538884/v1/0a6f995f-77e8-4fe9-86e0-9a77203b893a.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Toxoplasma gondii detection and genotyping in the Environmental matrices: soil and water in Gaza, Palestine","fulltext":[{"header":"Introduction","content":"\u003cp\u003e\u003cem\u003eT. gondii\u003c/em\u003e is an obligate intracellular protozoan replicating with a three-stage life cycle that is infectious to all warm-blooded animals(Robert-Gangneux and Dard\u0026eacute; \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Maenz et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).Sexual reproduction occurs exclusively in the feline definitive host, leading to the formation of oocysts that are excreted in their feces. After sporulation in the environment, oocysts (the environmental stage) become infective. \u003cem\u003eT. gondii\u003c/em\u003e oocysts are resistant to extreme environmental, physical, and chemical conditions and can survive for a long time in the environment (Robert-Gangneux and Dard\u0026eacute; \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Dum\u0026egrave;tre and Dard\u0026eacute; \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). Ingestion of sporulated oocysts by intermediate hosts results in the release of sporozoites, which pass through the intestinal epithelium and develop into tachyzoites. This is followed by tachyzoite spread through body fluids, including enormous and rapid asexual reproduction in nucleated cells of the host (Robert-Gangneux and Dard\u0026eacute; \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Maenz et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). This stage of infection is distinguished by a significant inflammatory response and host tissue damage, which can lead to clinical signs of toxoplasmosis. After the host's immunological response has progressed, replication slows, and the now-called bradyzoites settle in host cells of the retina, brain, skeletal, and cardiac muscles (Blanchard et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The parasite may avoid the immune system and lives a lifespan within the host by forming tissue cysts. Humans are susceptible to \u003cem\u003eT. gondii\u003c/em\u003einfection, which may be acquired horizontally by ingestion of oocysts via infected food (vegetables, fruits, and water), infection after gardening, or direct contact with cat feces. Consumption of raw or undercooked meat harboring highly infectious tissue cysts, can also result in oral-alimentary transmission)Tenter et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Dias et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). A Research conducted in 1999 showed that drinking water functioned as a vehicle for oocyst transmission among Jains, a vegetarian group in India (Hall et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). Besides, a large outbreak of toxoplasmosis was recorded in Brazil in 2001 and 2002 as a result of consuming water from municipal sources (De Moura et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Certainly, a variety of environmental conditions contribute to the spread of \u003cem\u003eT. gondii\u003c/em\u003e. These serve as a vector for the parasite's transmission to surface water sources, some of which include heavy rains and winds(Mac Kenzie et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e1994\u003c/span\u003e). Toxoplasmosis is the third most common food-borne disease, behind salmonellosis and listeriosis (Jones et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Dubey and Jones \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2008\u003c/span\u003e), as well as being one of the most widespread zoonotic diseases. It is believed that about 30% of the human population is infected with \u003cem\u003eT. gondii\u003c/em\u003e (Maenz et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). While the seroprevalence of this parasite is expected to be greater than 50% in regions where raw or undercooked meat is often consumed. Furthermore, high seroprevalence is prevalent in tropical parts of Latin America and Africa, where cat populations are considerable and climatic conditions are favorable for oocyst survival (Jones et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2001b\u003c/span\u003e; Di Carlo et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Dubey et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). It is estimated that 15% of childbearing women aged 25 to 44 years in the United States are infected with \u003cem\u003eT. gondii\u003c/em\u003e. Meanwhile, it is reported that between 400 and 4,000 cases of congenital toxoplasmosis occur annually (Jones et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2001b\u003c/span\u003e). \u003cem\u003eT. gondii\u003c/em\u003e was also ranked fourth in an assessment of the worldwide significance of 24 food-borne parasites based on socioeconomic and trade impacts(FAO/WHO \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Furthermore, occasional outbreaks of waterborne toxoplasmosis have been observed in various places of the world e.g., Panama, British Columbia, and Brazil (Benenson et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1982\u003c/span\u003e; Bowie et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e1997\u003c/span\u003e; Lass et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Despite the fact that contaminated drinks and foods constitute the significant risk factors, a variety of other factors, including poor socioeconomic and educational levels, do also contribute to the occurrence of toxoplasma outbreaks (Ferreira et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Numerous studies have been conducted to investigate the seroprevalence of \u003cem\u003eT. gondii\u003c/em\u003e infection in pregnant and abortion-experiencing women in Palestine. According to Al-Hindi and Lubbad (2009), the occurrence of \u003cem\u003eT. gondii\u003c/em\u003e specific antibodies was 12.8% for IgM among 312 women who had abortions in Gaza-Palestine (Al-Hindi and Lubbad 2009).While among 204 pregnant women in Hebron-Palestine, a high seroprevalence of 27.9% was recorded with women from rural areas being more affected than those from urban areas (Nijem and Al-Amleh \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Furthermore, in Gaza, Al-Jarousha (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), reported 30.9% seropositivity among 255 pregnant women (Al-Jarousha \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). The subject study's goal was to detect \u003cem\u003eT. gondii\u003c/em\u003e oocysts in environmental samples, such as soil and water, in Gaza, Palestine, using conventional (Sheather's solution) and molecular (PCR) techniques, as well as to determine the genotype of some of the isolates.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cp\u003e\u003cstrong\u003eStudy area and samples collection \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA total of 200 soil samples were randomly collected, with 40 samples taken from each of the following locations: Feras market, \u0026nbsp;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\u0026rsquo;s waste\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003ewater treatment plant as shown in table (1), below \u0026nbsp;which outlines the number of samples and the corresponding locations from which they were collected.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable\u0026nbsp;1.\u003c/strong\u003e\u0026nbsp; Type, source, and number of samples collected in the current investigation.\u003c/p\u003e\n\u003ctable border=\"1\" cellpadding=\"0\" cellspacing=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"38.745980707395496%\"\u003e\n \u003cp\u003e\u003cstrong\u003eLocality\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"35.69131832797428%\"\u003e\n \u003cp\u003e\u003cstrong\u003eType of sample\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25.562700964630224%\"\u003e\n \u003cp\u003e\u003cstrong\u003eNumber of samples\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"38.745980707395496%\"\u003e\n \u003cp\u003eWadi \u0026ndash; Gaza\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"35.69131832797428%\"\u003e\n \u003cp\u003eSoil\u003c/p\u003e\n \u003cp\u003eWater (Running)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25.562700964630224%\"\u003e\n \u003cp\u003e40\u003c/p\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"38.745980707395496%\"\u003e\n \u003cp\u003eTrash dumpster premises\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"35.69131832797428%\"\u003e\n \u003cp\u003eSoil\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25.562700964630224%\"\u003e\n \u003cp\u003e40\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"38.745980707395496%\"\u003e\n \u003cp\u003eHouses yards\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"35.69131832797428%\"\u003e\n \u003cp\u003eSoil\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25.562700964630224%\"\u003e\n \u003cp\u003e40\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"38.745980707395496%\"\u003e\n \u003cp\u003ePublic squares and gardens\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"35.69131832797428%\"\u003e\n \u003cp\u003eSoil\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25.562700964630224%\"\u003e\n \u003cp\u003e40\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"38.745980707395496%\"\u003e\n \u003cp\u003eFeras market\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"35.69131832797428%\"\u003e\n \u003cp\u003eSoil\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25.562700964630224%\"\u003e\n \u003cp\u003e40\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"38.745980707395496%\"\u003e\n \u003cp\u003eSewage treatment plant\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"35.69131832797428%\"\u003e\n \u003cp\u003eWater (Sewage)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25.562700964630224%\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"38.745980707395496%\"\u003e\n \u003cp\u003eWells\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"35.69131832797428%\"\u003e\n \u003cp\u003eWater (Household)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25.562700964630224%\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"38.745980707395496%\"\u003e\n \u003cp\u003eTotal soil samples\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"35.69131832797428%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25.562700964630224%\"\u003e\n \u003cp\u003e200\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"38.745980707395496%\"\u003e\n \u003cp\u003eTotal water samples\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"35.69131832797428%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25.562700964630224%\"\u003e\n \u003cp\u003e90\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"38.745980707395496%\"\u003e\n \u003cp\u003eTotal environmental samples\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"35.69131832797428%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25.562700964630224%\"\u003e\n \u003cp\u003e290\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003eWadi 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 \u0026nbsp;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.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetection of \u003cem\u003eT. gondii\u003c/em\u003e oocysts in soil and water:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFrom 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 \u0026mu;m 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\u0026apos;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\u0026apos;s surface layer and mixed with the sediment in order to be microscopically examined using the Sheather\u0026apos;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 B\u0026uuml;chner funnel with a 0.45 \u0026mu;m nylon filter membrane. Using saline, the membrane was washed and then examined through the light microscope employing Sheather\u0026rsquo;s solution. Contaminated samples were put into Eppendorf tubes and stored at -20 \u0026deg;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)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMolecular Identification and Genotyping of \u003cem\u003eT. gondii\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDNA Extraction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe 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 \u0026mu;L TE buffer (10 mMTris, 1 mM EDTA, pH 7.2) and preserved at -20 \u0026deg;C until the PCR procedure was completed (Amairia et al. 2016).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePCR Primers and Conditions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe PCR reaction was performed using a set of primers-TOXO1 (5\u0026apos; GGA ACT GCA TCC GTT CAT GAG 3\u0026apos;) and Toxo2 (5\u0026apos; TCT TTA AAG GGT TGG TGG TC 3\u0026apos;) reported by Lass \u003cem\u003eet al\u003c/em\u003e. (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\u0026apos; AGG GGA GGG TGA GGA TGA 3\u0026apos;) and TOXO-R (5\u0026apos; TGG TCT CGT CTG GAT CGC AT 3\u0026apos;) 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 \u0026deg;C, thermocycling for 30 cycles, with each cycle consisting of 30 seconds at 94 \u0026deg;C followed by 30 seconds at 55 \u0026deg;C for annealing, 45 seconds at 72 \u0026deg;C for extension, and a final extension cycle of 10 minutes at 72 \u0026deg;C (Lass et al. 2009; Hassanain et al. 2013; Dardona et al. \u0026nbsp;2021). Distilled water was used as a negative control, while a previously identified and well-defined \u003cem\u003eT. gondii\u003c/em\u003e 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.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGenotyping\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe 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 \u003cem\u003eMseI\u003c/em\u003e. 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. \u0026nbsp; 2016).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical Analysis\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAnalysis 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 \u003cem\u003eT. gondii\u003c/em\u003e 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.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eThe present study results revealed that \u003cem\u003eT. gondii\u003c/em\u003e oocysts had a conspicuous prevalence in some of the examined environmental samples. More precisely, 22 (7.58%) samples out of the 290 tested water and soil samples were found to be contaminated with oocysts according to the PCR technique, while 31 (10.68%) were positive according to Sheather\u0026rsquo;s solution. Of the PCR-positive samples 18 were soil samples and four water samples. In the case of Sheather's solution, 26 of the 31 contaminated samples were from soil and 5 were from water samples. The distribution and comparison of the data obtained from the two techniques employed in the study are shown in Table\u0026nbsp;(2) below. The current investigation concluded that water samples collected from Wadi Gaza were the only source of oocyst contaminated water, despite the fact that PCR and Sheather's solution revealed 4 and 5 contaminated samples, respectively. In addition, \u003cem\u003eT. gondii\u003c/em\u003e oocysts were not detected in samples collected from wells or the sewage treatment facility. On the other hand, contamination has been documented by every source of soil samples. The greatest rate of contamination was seen in samples acquired from residential home yards, with 9 and 7 samples detected by Sheather's and PCR, respectively. Feras market and Wadi Gaza ranked second and third in terms of the level of oocyst contamination, with 5 samples per Sheather's solution for both of them, and 5 and 3 samples per PCR, respectively. Following that, soil samples were collected from the premises of garbage dumpsters, with 4 and 2 samples, respectively, according to Sheather's and PCR. Lastly, samples collected from public gardens and squares had the lowest contamination rate, with two samples per Sheather's solution and one sample per PCR (Table\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). As shown in Table\u0026nbsp;(2), there is a significant difference in the incidence of \u003cem\u003eT. gondii\u003c/em\u003e oocysts in the various environmental samples studied by both techniques and their associated location sources. In terms of the month in which the samples were gathered, the subject study's findings showed an interesting trend. The highest number of contaminated samples was found in December, followed by November, October, September, August, and July (Table \u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The month of July had the lowest rate of contaminated samples.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePrevalence of \u003cem\u003eT. gondii\u003c/em\u003e oocysts in the environmental samples, based on the source of sample using both PCR and Sheather\u0026rsquo;s techniques.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEnvironmental Sample (Type)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSample (No)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePositive (Sheather\u0026rsquo;s)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePositive (PCR)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWadi \u0026ndash;Gaza (Running water)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSewage treatment plant (sewage water)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWells (drinking and irrigation)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal water samples\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5 (5.55%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4 (4.44%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWadi \u0026ndash; Gaza (Soil)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTrash dumpster premises(Soil)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHouse yards (Soil)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePublic gardens (Soil)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFeras market (Soil)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal soil samples\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e200\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e26 (13.00%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e18 (9.00%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal (Water and Soil)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e290\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e22\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e% positive\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10.68%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7.58%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"4\" nameend=\"c4\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eχ2 (PCR)\u003c/b\u003e\u0026thinsp;=\u0026thinsp;\u003cb\u003e16.79 P\u0026thinsp;=\u0026thinsp;0.01 P\u0026nbsp;\u0026lt; 0.05. Statistically significant\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003eχ2 (Sheather\u0026rsquo;s ) = 14.86 P = 00.038 P\u0026nbsp;\u0026lt; 0.05. Statistically significant\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe relationship between sample contamination with oocysts by both techniques and the months in which samples were collected had demonstrated no statistical significance, as indicated by the findings in table (3) below. Figures\u0026nbsp;(2) below presents a photograph of ethidium bromide stained agarose gel illustrating some of the PCR-positive \u003cem\u003eT. gondii\u003c/em\u003e oocyst samples.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMonthly prevalence of \u003cem\u003eToxoplasma gondii\u003c/em\u003e oocyst in the environmental samples in Gaza city during the period July-December 2019.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMonth\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSamples (No)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSoil (No)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eWater (No)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e(+\u0026thinsp;Ve) Sheather\u0026rsquo;s\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e(+\u0026thinsp;Ve) PCR\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eJuly\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAugust\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSeptember\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOctober\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNovember\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDecember\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e290\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e200\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e31 (10.68%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e22 (7.58%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"6\" nameend=\"c6\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eχ2\u003c/b\u003e ( \u003cb\u003ePCR) = 9.047 P = 00.107 P\u0026nbsp;\u0026gt; 0.05. Not Statistically significant\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003eχ2 (Sheather\u0026rsquo;s ) = 8.600 P = 00.126 P\u0026nbsp;\u0026gt; 0.05. Not Statistically significant\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eRegarding the PCR-RFLP genotyping, figure (3) below shows that the \u003cem\u003eMseI\u003c/em\u003e digestion products are 544 and 168 bp thus indicating that the four samples tested are of Toxo-type I.\u003c/p\u003e "},{"header":"Discussion","content":"\u003cp\u003eThe study's main purpose was to detect \u003cem\u003eT. gondii\u003c/em\u003e oocysts in environmental samples, particularly soil and water. Given that the majority of \u003cem\u003eT. gondii\u003c/em\u003e infections in pregnant women occur as a result of soil contact, as soil is assumed to be an important environmental factor in human infections (Ajmal et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). According to a European research, 17% of \u003cem\u003eT. gondii\u003c/em\u003e infections are caused by contact with contaminated soil, as demonstrated by a direct survey of women infected with the parasite. Because oocysts can remain infectious in soil for more than a year, contaminated soil is seen as a serious risk factor in the parasite's spread (Lass et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Indeed, soil is an important environmental source of \u003cem\u003eT. gondii\u003c/em\u003e infection in humans and animals (Cook et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; dos Santos et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). This is corroborated by the findings of the present investigation, which revealed the existence of oocysts in all of the locations where soil samples were taken. Because oocysts are exclusively excreted through cat feces, and every site where samples were taken included cats. Because stray cats are found in almost every area of Gaza, particularly in Wadi Gaza and the surrounding agricultural lands, where there are abundant food sources and this explains the highest rate of contamination detected in Wadi-Gaza and residential home yards. Many of the cats found in those areas tend to be infected with \u003cem\u003eT. gondii e.g.\u003c/em\u003e, Al-Hindi et al (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). documented the occurrence of \u003cem\u003eT. gondii\u003c/em\u003e oocysts in the feces of some stray cats in a study conducted in Khanyounis Governorate, located in the south of Gaza City (Al-Hindi et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Since they are stray cats, they may easily move from one region to another, making them significant risk factors for \u003cem\u003eT. gondii\u003c/em\u003e oocyst transmission and contamination of soil and flowing water. This is understandable considering that cats, as previously stated, play a role in the dissemination of oocyst. The results of the current study are consistent with those of a comparable study conducted in Poland, which showed the existence of oocysts in 18 (17.8%) of 101 soil samples using the PCR technique. Furthermore, another study conducted in China reported that 34 (12.69%) of 286 soil samples were contaminated with \u003cem\u003eT. gondii\u003c/em\u003e oocysts (Lass et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Du et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). The present study's findings revealed that the surface water stream, Wadi Gaza, was contaminated with oocyst. This water stream is a mix of rain and sewage water that flows from east to west across Gaza and into the Mediterranean Sea. The presence of cats beside the water stream can be blamed for the contamination of this water. This creek provides nutritional resources to cats due to people's terrible habit of dumping waste along the stream, as well as the existence of local and immigrant birds, rats, and decaying animal corpses. Besides, other environmental elements that impact the spread of oocysts in surface water sources include rain, wind, soil, and mechanical vectors such as insects, flies, and earthworms (Dum\u0026egrave;tre and Dard\u0026eacute; \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). Other water samples collected from other locations were found to be free of oocyst contamination. This can be attributed to the absence of infected cats and other variables previously identified as being important for oocyst dissemination. The wells and the water treatment plant were correctly closed to avoid the existence of cats or the effect of other factors capable of spreading these oocysts. Another study conducted in the study area regarding the detection of parasitic contamination in the water used for kitchen purposes and carried out by AL-Hindi et al. (2021) confirms the results of the current study, as their study did not record the occurrence of \u003cem\u003eT. gondii\u003c/em\u003e oocysts in this water and only recorded the occurrence of \u003cem\u003ecryptosporidium\u003c/em\u003e (AL-Hindi et al. 2021). It is also commonly known that the water utilized for household purposes in that area is sourced from wells distributed around the area. On the other hand, \u003cem\u003eT. gondii\u003c/em\u003e was not regarded as a serious aquatic pathogen, in contrast to \u003cem\u003ecryptosporidium\u003c/em\u003e (Mac Kenzie et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e1995\u003c/span\u003e). In view of current toxoplasmosis outbreaks in people, the topic study emphasized the significance of oocyst contamination in environmental samples. The findings of the current study were comparable to those of a study conducted in France, where oocysts were found in 37 (7.7%) of 482 environmental water samples (Aubert and Villena \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). According to the interpretations formed by the author of the French study based on the study's data, the major cause of this contaminated water was contaminated soil rinsed by rain water. This view, in fact, confirms the conclusion obtained from the subject study's findings. Rainwater combined with dirt and sewage may have played a significant part in the oocyst contamination of Wadi Gaza, the primary surface water stream that runs for a long distance before emptying into the Mediterranean. To investigate the authenticity of this phenomenon, water samples from Wadi Gaza were obtained just one kilometer before its endpoint, the Mediterranean Sea. Given the length of the stream, this collection point enabled the subject researchers to recognize and quickly investigate the potential impact of soil and other environmental elements on water contamination. Not to mention, the topic investigation indicated a congruent conclusion based on comparable results obtained from a study conducted in Brazil, which discovered 3 (7.7%) of 39 environmental water samples collected from surface water streams were utilized largely for drinking water (Galvani et al. 2019). This research, as well as the previous ones, corroborates the existence of oocyst contamination in surface water sources, which is regarded as a high contamination rate, particularly when utilized for irrigation. The current investigation is one of the few that have been conducted to detect oocyst contamination or even parasites in the Gaza environment. In their study, Dardona et al. (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) recorded the occurrence of \u003cem\u003eT. gondii\u003c/em\u003e oocysts in raw leafy vegetables, which are commonly consumed raw in Gaza. One of their interpretations of their findings is that soil or water contamination, as well as some environmental factors, play a significant role in oocyst spread to vegetables (Dardona et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Another study conducted in the study region by Hilles et al. (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), recorded the contamination of the Gaza shoreline with certain parasites without mentioning \u003cem\u003eT. gondii\u003c/em\u003e among the detected parasites (Hilles et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Nonetheless, the small differences in the results obtained by different research conducted in different cities and countries might be attributable to a variety of factors. First, there are the various detection procedures used; second, there are the various environmental elements that aid in the transmission of oocysts; and finally, there is a variation in cat population rates and presence in the neighborhood of where the samples were gathered. Even before 1970, when the \u003cem\u003eT. gondii\u003c/em\u003e life cycle was established, there was a disagreement over the relevance of the epidemiological components of \u003cem\u003eT. gondii\u003c/em\u003e infection, whether by ingestion of oocysts or tissue cysts (Jackson and Hutchison \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e1989\u003c/span\u003e). Although the relationship between the contamination of soil and water samples and the month in which the samples were collected was not found to be statistically significant, it can be noted that the highest contamination rates were during the months of November and December, and the lowest levels of contamination were during the months of July and August. This is easily explained by the fact that the quantity of rain that falls during the winter raises the average water level in the valley's stream, increasing the speed of the valley's run-off, which transports possible sources of contamination. Furthermore, the high level of the stream causes flooding, and the water floods the agricultural lands located on the valley's boundaries. This further increases the rate of contamination. Furthermore, the possibility of the previously stated environmental factors, such as rain water and wind, in the transmission and dissemination of \u003cem\u003eToxoplasma\u003c/em\u003e oocysts rises during the winter (Dum\u0026egrave;tre and Dard\u0026eacute;. 2003). The first observation made upon discovery was that both vegetarians and non-vegetarians are susceptible to \u003cem\u003eT. gondii\u003c/em\u003e infections, suggesting that carnivorism was not the only source of \u003cem\u003eT. gondii\u003c/em\u003e infections (Dubey \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Shapiro et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The degree of \u003cem\u003eT. gondii\u003c/em\u003e infection in cats is determined in conjunction with the degree of infection in birds, rodents, and other sources of cat food. Just as the incidence of oocysts in the environment is increasing, contamination of surface water leading to sea water constitutes a concerning issue (Sibley and Boothroyd \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e1992\u003c/span\u003e; Afonso et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Infected cats defecate millions of oocysts in soil after swallowing only one tissue cyst or bradyzoite, and the sporulated oocysts stay infectious in soil for up to 18 months, independent of temperature exposure. Despite this, finding oocysts with a light microscope was sufficient for significantly contaminated samples. However, the molecular technique is more tempting because of its high sensitivity and rapidity in detecting \u003cem\u003eT. gondii\u003c/em\u003e by PCR amplification of the B1 gene (Dum\u0026egrave;tre and Dard\u0026eacute;, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Dubey, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2009\u003c/span\u003e).The current study, on the other hand, employed a PCR-RFLP assay on the GRA6 gene to genotype four DNA isolates. It is worth noting that this is the first study in the research area that tackled the genotyping of \u003cem\u003eT. gondii\u003c/em\u003e isolates, and GRA6 was used in the parasite genotyping process because it is a coding region with a larger and more diverse polymorphism than other regions such as SAG1, SAG2, and GRA4 (Norouzi et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Furthermore, the current study's findings revealed that all of the analyzed oocyst DNA isolates belonged to the Toxo-Type I. The genotyping of the parasite is regarded as a critical component in generating the disease, as demonstrated by several mouse model trials, and it has been documented in greater detail that genotype I strains are very virulent, but the other kinds are not harmful to a considerable extent (Sibley and Boothroyd \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e1992\u003c/span\u003e.). It is crucial to note that \u003cem\u003eT. gondii\u003c/em\u003e DNA isolates were categorized based on their virulence in outbred mice, but the first phylogenetic investigations of \u003cem\u003eT. gondii\u003c/em\u003e strains revealed that their complexity was significantly lower than predicted (Dard\u0026eacute; et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1992\u003c/span\u003e; Sibley et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e1992\u003c/span\u003e). One of the most essential aspects of genotyping is its application in tracking epidemics (Ajzenberg et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). It has been demonstrated that there are large geographical variations in the distribution of \u003cem\u003eT. gondii\u003c/em\u003e genotypes. For example, certain investigations performed in Spain and Portugal have reported the existence of types I and III in these locations (Fuentes et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; de Sousa et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). On the other hand, the findings of investigations conducted to assess the genotyping of \u003cem\u003eT. gondii\u003c/em\u003e for isolates in both Crete and Cyprus reported the existence of type III in both areas (Messaritakis et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). To round out the findings, French research revealed that only four of 86 \u003cem\u003eT. gondii\u003c/em\u003e isolates belonged to genotype I. On the contrary, genotype I was shown to be extremely dominant in one of the investigations done on the isolated strains of CSF of eight patients with acquired immunodeficiency in the United States, as the findings of this study revealed that the majority of them were infected with \u003cem\u003eT. gondii\u003c/em\u003e genotype I (Ajzenberg et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Khan et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). On the other hand, in a study conducted in Brazil to identify the genotyping of \u003cem\u003eT. gondii\u003c/em\u003e isolates reported in water supply samples, after genotyping them based on the SAG2 locus, it was reported that genotype I is very pathogenic (De Moura et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2006\u003c/span\u003e).\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe current study concluded that \u003cem\u003eT. gondii\u003c/em\u003e oocyst contamination in soil is greater than in water. The highest rate of soil contamination was detected in samples obtained from private home yards, whereas the lowest rate was reported in public squares and gardens. The contaminated water samples, on the other hand, are solely from flowing water samples gathered from Wadi Gaza, whereas water collected from other sources was confirmed to be clear of contamination. Finally, this investigation revealed that all genotyped samples belonged to \u003cem\u003eT. gondii\u003c/em\u003e type I.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to express their thanks and gratitude to Mr. Muhammad Ashour for his technical assistance in the PCR work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors affirm that the data supporting this study are available within the article and that raw data supporting the study\u0026apos;s findings are available from the first author, Zuhair Dardona, upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e1- Zuhair Dardona ( Practical work\u0026ndash; writing \u0026ndash; statically analysis).\u003c/p\u003e\n\u003cp\u003e2-Adnan Al-Hindi ( writing \u0026ndash; review)\u003c/p\u003e\n\u003cp\u003e3- Samia boussaa \u0026nbsp;( writing- review)\u003c/p\u003e\n\u003cp\u003e4- Mohamed Hafidi (Review)\u003c/p\u003e\n\u003cp\u003e5- Ali boumezzough (Reviw)\u003c/p\u003e\n\u003cp\u003e6- Fadel \u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003eSharif ( Genotyping \u0026ndash; review)\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOn August \u0026nbsp;5, 2019, the ethical research committee of the Islamic University of Gaza approved an ethical clearance for this study, which planned to gather water and soil samples from the study area.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll of the authors have willingly agreed to take part in this research project.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors consent to the publishing of identifying facts, which may include photographs, details within the text (\u0026quot;Material\u0026quot;) data, and everything inside this paper, in a parasitology research journal.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAfonso E, Thulliez P, Pontier D, Gilot-Fromont E (2007) Toxoplasmosis in prey species and consequences for prevalence in feral cats: not all prey species are equal. \u003cem\u003eParasitol\u003c/em\u003e 13414:1963\u0026ndash;1971.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAjmal A, Maqbool A, Qamar MF, Ashraf K, Anjum AA (2013) Detection of \u003cem\u003eToxoplasma gondii\u003c/em\u003e in environmental matrices (water, soil, fruits and vegetables). \u003cem\u003eAfr J Microbiol Res\u003c/em\u003e 716: 1505\u0026ndash;1511.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAjzenberg D, Banuls AL, Su C, Dumetre A, Demar M, Carme B, Dard\u0026eacute; ML(2004) Genetic diversity, clonality and sexuality in \u003cem\u003eToxoplasma gondii\u003c/em\u003e. \u003cem\u003eInt J Parasitol\u003c/em\u003e 3410:1185\u0026ndash;1196.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAl Hindi I A, Lubbad A M (2009) Seroprevalence of toxoplasmosis among Palestinian aborted women in Gaza. \u003cem\u003eAnn Alquds Med\u003c/em\u003e 5:39\u0026ndash;47.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAl-Hindi AI, Abu-Draz M, El-Zenati A, Ali AA, Dagga AA. 2019. 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For this purpose290 environmental samples, including 200 from soil and 90 from water, were collected. Soil samples were collected from Wadi-Gaza, vicinity of trash dumpsters, residential home yards, open-air marketplaces, public squares and gardens. Water samples were collected from Wadi-Gaza, wells used for drinking and irrigation. The samples were examined utilizing PCR and Sheather's solution techniques and genotyping of 4 samples was performed by PCR-RFLP. The results revealed that out of the 290 investigated samples, 31 (10.68%) proved contaminated by Sheather\u0026rsquo;s solution, whereas 22 (7.58%) were positive by PCR. Genotyping results showed that all of the four tested samples belonged to \u003cem\u003eT. gondii\u003c/em\u003e Type I. The highest rate of contamination was detected in the samples collected from residential house yards, while public squares and gardens represented the lowest rate. Regarding the water samples, \u003cem\u003eT. gondii\u003c/em\u003e oocysts were detected only in the samples collected from Wadi-Gaza. This work confirms the presence of Type I \u003cem\u003eT. gondii\u003c/em\u003e in the soil and water of the study area, with soil contamination being heavier than that of water.\u003c/p\u003e","manuscriptTitle":"Toxoplasma gondii detection and genotyping in the Environmental matrices: soil and water in Gaza, Palestine","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-04-15 19:58:00","doi":"10.21203/rs.3.rs-1538884/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"8530f4db-5041-4e1e-b332-466f05fbab3b","owner":[],"postedDate":"April 15th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2022-05-09T03:44:05+00:00","versionOfRecord":[],"versionCreatedAt":"2022-04-15 19:58:00","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-1538884","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1538884","identity":"rs-1538884","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}