Detection of Toxoplasma gondii oocysts in contaminated soils using polymerase chain reaction analysis in Duhok City Iraq

Arshad Mohammad Abdullah; Narmin Saeed Merza; Ahmed Saeed Merza

doi:10.4103/MJBL.MJBL_29_22

ORIGINAL ARTICLE

Year : 2022 | Volume : 19 | Issue : 2 | Page : 271-274

Detection of Toxoplasma gondii oocysts in contaminated soils using polymerase chain reaction analysis in Duhok City Iraq

Arshad Mohammad Abdullah¹, Narmin Saeed Merza², Ahmed Saeed Merza³
¹ Department of Basic Science, College of Pharmacy, University of Duhok, Duhok, Iraq
² Scientific Research Center, College of Science, University of Duhok, Duhok, Iraq
³ Department of Biology, College of Science, University of Duhok, Duhok, Iraq

Date of Submission	05-Feb-2022
Date of Acceptance	20-Feb-2022
Date of Web Publication	30-Jun-2022

Correspondence Address:
Arshad Mohammad Abdullah
Department of Pharmacology, College of Pharmacy, University of Duhok, Iraqi Kurdistan, Duhok City
Iraq

Source of Support: None, Conflict of Interest: None

DOI: 10.4103/MJBL.MJBL_29_22

Abstract

Background: The impact of the acquisition of Toxoplasmosis from the environment through ingestion of oocyst from the soil, food, or water contamination remains worrisome worldwide. Objective: This study aimed to apply the molecular method for the detection of the occurrence of Toxoplasma oocysts by polymerase chain reaction (PCR) from different soil samples in Duhok province, Kurdistan region of Iraq. Materials and Methods: In this study, the occurrence of soil contamination with Toxoplasma gondii oocysts was recorded in different Duhok City’s areas by collecting 144 soil samples from different places. Results: Only seven samples (4.86%) were identified as T. gondii oocysts using microscopical identification and confirmed at the molecular level via PCR analysis using a fragment of 122 bp as a target of B1 gene. No significant difference was observed among soil types; parks showed the highest percentage 2.77%, followed by playgrounds (1.38%), whereas rubbish dumps accounted for 0.96%. Conclusion: The current study showed, a low percentage of T. gondii oocysts are recorded in soil samples as compared with other studies worldwide, but there is still the risk of infection among people who ramble at these places remains, especially among children who play with soil at these places.

Keywords: Detection, PCR, Toxoplasma gondii oocysts, toxoplasmosis

How to cite this article:
Abdullah AM, Merza NS, Merza AS. Detection of Toxoplasma gondii oocysts in contaminated soils using polymerase chain reaction analysis in Duhok City Iraq. Med J Babylon 2022;19:271-4

How to cite this URL:
Abdullah AM, Merza NS, Merza AS. Detection of Toxoplasma gondii oocysts in contaminated soils using polymerase chain reaction analysis in Duhok City Iraq. Med J Babylon [serial online] 2022 [cited 2023 May 29];19:271-4. Available from: https://www.medjbabylon.org/text.asp?2022/19/2/271/349481

Introduction

Toxoplasmosis is a global zoonotic disease caused by the Toxoplasma gondii parasite that infects humans and other warm-blooded animals.^[1] It has been estimated that approximately 30% of humans are infected by this parasite.^[2],[3] Although T. gondii infections are usually asymptomatic, they can cause severe infections, especially in congenital cases and immunocompromised patients.^[4],[5],[6] Humans are infected with Toxoplasmosis primarily by oral ingestion of viable cysts present in raw or undercooked infected meat and ingestion of oocyst in water, soil, vegetable, and fruits that are contaminated with stool of infected cats.^[7],[8],[9] In toxoplasmosis, infected stray cats play an important role in the epidemiology of this parasite through shedding the infective stage (Oocysts) into the environment. Toxoplasma gondii oocysts have the ability to be resistant to unfavorable environmental conditions since they maintain their infectivity for more than 12 months in soil and about 54 months in water.^{[10],[11],[12]} Thus, soil and water play a vital role in the transmission of the infective stage and act as sources of T. gondii infection in humans.^[13],[14] Detection of T. gondii oocysts in environmental samples is a great challenge as this coccidian parasite could be responsible for severe infections in humans and animals via ingestion of a single oocyst from contaminated water, soil, fruits, or vegetables.^[12] Documentation of the incidence of soil contamination with T. gondii oocyst plays a crucial role in minimizing toxoplasmosis infection.^[15] However, there is no sufficient information concerning T. gondii oocysts prevalence in the environment, especially in soil.^[16] Diagnostic researches and environmental samples examination are more realizable by using recent specific, sensitive, and developed methods such as molecular techniques to detect and identification of Toxoplasma oocysts..^{[12],[17],[18],[19]} Therefore, this study aimed to apply the molecular method for the detection of the occurrence of Toxoplasma oocysts in different soil samples in Duhok province, Kurdistan region of Iraq.

Materials and Methods

Study area

Duhok province is located in the northwest of Iraq and surrounded on three sides by mountain ranges; the city mostly contains hills, mountain slopes, and valleys with the plain area on the west. Duhok city climate is very hot and dry in summer and mild too cold in winter.

Samples collection

One hundred forty-four soil samples were collected from different places in Duhok province, Kurdistan region of Iraq in a period between May and July 2021. Samples were collected from different public areas such as playgrounds, parks, rubbish dumps, and areas where cats may excrete oocysts. In this study, special procedures were used in soil sample preparation as follow: From the surface layer of ground in aforementioned areas, 300 g of soil were obtained from a depth of 3–5 cm, and all soil samples were dried for 2 days at room temperature, and after sieve of soil samples, 30 g of each sample were taken and were used in further examinations.^[20]

Oocyst recovery

In order to recover oocysts of the parasite from the collected soil samples, the floatation method was used according to Matsuo et al.,^[21] with minor modification. In this method, 30 g of filtered soil samples through two layers of gauze re-suspended with 40 mL of 0.1% Tween 80 solution. The sample was distributed into six (10 mL) tubes, centrifuged at 3000 rpm for 10 min, the supernatants were discarded, then the sediment of each tube was re-suspended in 6 mL of sucrose solution (specific gravity 1.200) and centrifuged at 3000 rpm for 10 min. The supernatant of each tube transferred into another clean 10 mL tube and diluted with distal water (1:10), centrifuged as described in the above step. The obtained sediments of each tube were collected into a new tube, mixed well, and some of sediment was kept for DNA extraction for those samples which giving positive detection of T. gondii oocysts after microscopic examination. The other amount of sediment was re-suspended with 8 mL of flotation solution, centrifuged at 1000 rpm for 5 min. The tube was filled by syringe with flotation solution until reached to the brim; the coverslip was placed on the mouth of the tube and directly put on the glass slide for microscopically examination at (×200).^[21]

DNA extraction and specific detection of Toxoplasma gondii by polymerase chain reaction technique

Genomic DNA of all samples was extracted using a commercial Genomic Mini Kit (Bioneer, South Korea) according to manufacturers’ instructions. All obtained DNA was stored at –20°C. To detect oocysts of T. gondii, conventional polymerase chain reaction (PCR) analysis was used targeting fragment with 122 bp of B1 gene, with the primers, 5′-AAC GGG CGA GTA GCA CCT GAG GAG A 3′, and reverse primer, 5′-TGG GTC TAC GTC GAT GGC ATG ACA AC 3′ as described by Solymane et al.^[22] The amplification mixture consisted of 12.5 μL of reaction mix (GoTaq Master Mix/ Promega, Cat. No. M7822), 1 μL of each primer (10 pmol), 0.5 μL of 10% BSA (to remove PCR inhibitors), and 2.5 μL of soil template DNA in a final volume of 25 μL. The amplification process was as follows: 5 min at 94°C initial steps, followed by 35 cycles: denaturation at 95°C, for 30 s, 30 s for annealing at 58°C, and 30 s for the extension at 72°C; the final extension was at 72°C for 5 min. Electrophoresis analyzed PCR product on 1.5% agarose gel and staining process with ethidium bromide (2 μg/mL).

Statistical analyses

Prevalence of Toxoplasma gondii in different types of soil samples was analyzed by chi-square test by SPSS software program, version 21. A value of P < 0.05 was considered statistically significant.

Ethical considerations

The study was conducted in accordance with the ethical principles that have their origin in the Declaration of Helsinki. The research proposal was reviewed by the Scientific/Ethics committee and all the conditions of the research are accepted by the scientific committee, taking into consideration the following mandates: This article does not contain any studies with human or animal participants, and only soil samples were collected and used for the current study (reference no. 354, dated May 2, 2021).

Results

From a total of 144 soil samples obtained from different public areas in Duhok city, only 7 isolates were identified as oocyst of T. gondii based on morphological characteristics of oocyst of this parasite after microscopical identification. Parks represented the most contaminated region with T. gondii oocysts recording four positive samples from 48 soil samples, whereas 2 of 50 samples oocysts of this parasite were identified from playgrounds soil samples; only 1 of 46 samples was recovered from the rubbish dump [Figure 1]. However, no significant statistical differences in T. gondii were observed in different types of soil samples (P = 0.398) [Table 1].

Figure 1: Percentages of Toxoplasma gondii in different soil samples (No. 144) in Duhok city, Iraq

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Table 1: Prevalence of Toxoplasma gondii in different types of soil samples (No. 144) in Duhok city, Iraq

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These suspected isolates as T. gondii oocysts were subjected to PCR analysis to confirm it at the molecular level. All suspected samples were successfully amplified with molecular weight 122 bp as a fragment of B1 gene as the specific gene for detection of T. gondii as shown in [Figure 2].

Figure 2: PCR amplification of B1 gene of Toxoplasma gondii with amplicon molecular weight 122 bp. Electrophoresis was performed on (1.5%) agarose gel and run with 5V/cm, for 2 h. Lanes M contained DNA molecular weight marker (50 bp)

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Discussion

This study is concerned with the detection of T. gondii oocysts from the soil in Duhok City using microscopical identification and confirmed by the molecular method using genus-specific PCR analysis; a fragment with 122 bp of B1 gene was used as a target for identification. Seven isolates of T. gondii oocysts were recovered from over 144 soil samples collected from public areas in Duhok city accounting for 4.86%. Parks recorded 2.77% of soil contamination with T. gondii oocysts, playgrounds got 1.38% percentage of contamination, and only one isolate was collected from rubbish dumps accounting for 0.69%. No significant differences were found among soil types. To the best of our knowledge, this was the first study concerned with the detection of T. gondii oocysts from the environment in the Kurdistan region/Iraq. A comparative study on soil contamination with T. gondii oocysts between Baghdad and Kut cities found that a remarkable percentage of soil contamination was recorded in Baghdad city in the gardens (21.4%), schools (17.4%), and backyards of home (16.6%) compared to the low percentage of contamination (6%, 5.5%, 4%), respectively, observed in Kut city.^[23] Another study conducted in Iran found that the soil of public places was contaminated with oocyst of T. gondii in 8.66%.^[24] Overall, the reported prevalence of T. gondii in soil from various locations ranged from 0% (Hawaii, USA) to nearly 50% (Northeastern France).^[25] Many studies successfully applied PCR analysis for the detection of oocysts of this parasite.^{[12],[22],[24],[26],[27]} However, many factors may affect the results accuracy of T. gondii oocysts detection in the soil such as a low number of oocysts in the soil,^[12] confusion in the morphological differentiation of T. gondii oocysts, with other parasites oocysts, such as Neospora sp., Cryptosporidium, and Hammondia sp.,^[28] and hardness structure of T. gondii oocysts wall, sometimes cause difficult DNA extraction, especially in the conventional procedure.^[24] Furthermore, the presence of inhibitors in soil may affect the extraction of DNA as well as PCR reaction.^[29],[30] To ensure accuracy, commercial kits were used for the extraction of DNA and PCR reactions. Indeed, for extraction of nucleic acids and PCR technique, commercial kits were manufactured within such considerations to the removal of PCR inhibitors and increasing robustness of PCR enzymes.^[29] Ingestion of T. gondii oocysts in the soil is a significant transmission route for humans and animals acquiring toxoplasmosis.^[26] However, the detection of T. gondii oocysts contaminated soil of parks and playgrounds in Duhok city even though in low percentage alarm of the riskiness among people who hung out at these places especially children who usually play in direct contact with soil make them more vulnerable to infection. The soil of public parks contaminated with T. gondii oocysts may play a role in the epidemiology of toxoplasmosis and management of free-roaming cats as the main source of contamination and effective preventive measures should be considered.

Conclusion

The results of this study show contamination of soil with T. gondii oocyst, which may act as the source of acquiring toxoplasmosis. The results also suggest the application of molecular techniques for the detection and characterization of different species of coccidian parasites that may present in the soil in local areas and cause infections to humans as well as animals.

Data availability statement

All datasets generated or analyzed during this study are included in the manuscript.

Acknowledgement

Thanks are given to all laboratory staff for their efforts in this study.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

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