Molecular Prevalence of Toxoplasma Gondii in Butchers and Slaughtered Cattle in Middle Euphrates, Iraq

Introduction
Toxoplasma gondii is the most common zoonotic worldwide obligate intracellular parasite infecting humans via warm-blooded animals (Khalil et al., 2018). Wild and domesticated felines are the final hosts of T. gondii that sexually develop in the intestine, and the oocysts shed in the feces (Sakban et al., 2020; Jilo et al., 2021). Toxoplasmosis, a clinical disease caused by T. gondii, can probably infect all warm-blooded animals and humans and lead to several clinical symptoms, including fever, mental retardation, chorioretinitis, encephalitis, hydrocephalus in children, and abortion in livestock; however, the infection can lead to even mortality (Abdallah et al., 2019; Fazel et al., 2021; Khattab et al., 2022).
Although cattle infection is asymptomatic and leads to clinical signs or abortion, it plays a vital role in the transmission of toxoplasmosis to humans by the consumption of undercooked meat or unpasteurized milk because this parasite exits in the bloodstream and develops as tissue cysts in the muscles and glands (Opsteegh et al., 2020; Taalay et al., 2022).
There are several diagnostic methods used to identify T. gondii: Direct methods, such as preparation of blood smears from an infected host, histopathological examination and serological (immunological) methods, such as latex agglutination test, modified agglutination test, and enzyme-linked immunosorbent assay (Abdul Ameer Jaber & Noori, 2021; Liyanage et al., 2021). Different molecular techniques and real-time polymerase chain reaction (RT-PCR) methods have been developed to identify T. gondii using different samples, such as tissue biopsy and blood (Mahittikorn et al., 2005; Aghwan et al., 2021). Real-time PCR is a highly sensitive and specific method that is more accurate but costly and requires specialized detection systems (Santoro et al., 2019; Adriaanse et al., 2020; Souza et al., 2023).
Our study aimed to detect the incidence of toxoplasmosis in butchers and butchered cattle using the B1 gene and demonstrate the role of cattle in the zoonotic transmission of infection to humans.

Materials and Methods
A total of 100 blood samples were collected (2-3 mL) from butchers (male) and cattle (both sexes) of different age groups from different abattoirs during the period from September 1st, 2022, to September 30th, 2023. The blood samples were collected in an EDTA tube with anticoagulant and stored at -20°C according to the manufacturer’s instructions till used for DNA extraction.

Real-time PCR amplification
DNA extraction from blood samples using a Genomic DNA Mini Kit (Geneaid USA). Real-time PCR was done to detect T. gondii in blood samples using the primers and TaqMan probe for the B1 gene. This method was done based on the technique designated by Lin et al. (2000), quantitative PCR master mix existed organized using NEXpro™ qPCR Master Mix (Probe). The master mix was prepared following instructions of the company as follows: DNA template 5 µL, Forward B1 primer (5′-TCCCCTCTGCTGGCGAAAAGT-3′) (10 pmol) 1 µL, Reverse B1 primer (5′-AGCGTTCGTGGTCAACTATCGATTG-3′) (10 pmol) 1 µL (product size 94 bp), B1 probe (5′- FAM-TCTGTGCAACTTTGGTGTATTCGCAG-TAMRA- 3′) (20 pmol) 1 µL, quantitative PCR master PCR mix 10 µL and PCR water 2 µL. Subsequently, PCR master mix components were transported into an ExiSpin vortex centrifuge at 3000 rpm for 3 min. RT-PCR thermocycler situations were fixed according to the annealing temperature of primer and RT-PCR TaqMan kit instructions by BioRad thermocycler system of real-time PCR as in the follows: One cycle pre-denaturation 5 min in 95°C, denaturation 20 s in 95°C in 45 cycles, annealing extension 30 s in 60°C in 45 cycles and revealing for 30 s in 60°C in 45 cycles. The data analysis was performed using the threshold series number (CT value) calculation, which obtained the positive amplification of the T. gondii B1 gene in real-time PCR cycle numbers (Lin et al., 2000).

Statistical analysis
Computerized statistical analyses were performed using SPSS software, version 31. The chi-square test was employed to evaluate the variables (Joda, 2008).

Results
Total infection rates in butchers and cattle were 28% and 12%, respectively, out of 100 blood samples from each one, examined using real-time PCR (Table 1; Figures 1and 2). 

The infection rate of toxoplasmosis in butchers and cattle concerning age groups
Butchers 18-30 years old had the highest percentage (34.21%), followed by the age group 30-40 years (27.50%). In contrast, the lowest (18.18%) belonged to the age group <40 years without significant (P>0.05) difference (Table 2).

The age group 2-4 years of cattle recorded the highest infection rate (14.28%), and the lowest rate was recorded at the age group <1-2 years (10%) without significant (P>0.05) difference (Table 3). 

The infection rates of toxoplasmosis in cattle regarding the sex
Males had the highest infection rate (14.24%) compared to females (8.10%), with a significant difference (P>0.05) (Table 4).

Discussion
Toxoplasmosis is a common zoonotic disease worldwide caused by intracellular protozoan T. gondii, infecting a wide range of warm-blooded animals. The parasite can infect humans horizontally when consuming the infective stage (sporulated oocyst) in contaminated food and water or unpasteurized milk, undercooked, or meat containing bradyzoites (tissue cysts) of T. gondii. So cattle meat is an essential source of human infection, especially meat handlers (butchers). 
Real-time PCR is a highly accurate and suitable technique for diagnosis, providing a rapid, high sensitivity, specificity, and quantitative pathway for the detection of T. gondii infection in clinical specimens without false-positive results in comparison with serological tests (Awad & AL-Muffti, 2020; Adriaanse et al., 2020; Souza et al., 2023).
The overall prevalence found in butchers agreed with the results of Al-Khafaji (2014), who found 29.43% in meat cookers and sellers, as they frequently possess high-risk factors with parasites. However, it disagrees with Ibrahim et al. (2017), who mentioned 57.37% by real-time PCR for women consumed under-cooked meat, and Ebrahimzadeh et al. (2018), who recorded 68.47% in humans by nested PCR using the B1 gene. Ali et al. (2017) reported a lower infection rate of 0.62% in Saudi Arabia using the PCR B1 gene, while Al-Hadraawy & Hadi (2017) recorded 20.28% using the PCR B1 gene in Al-Najaf governorate, Iraq. Also, Khan et al. (2020) reported a lower infection rate of 20.64% in Pakistani males. These variations should be due to the differences in eating habits, method of cooking, kind of meat, social, economic, cultural habits, and sanitation coverage.
The highest percentage belonged to the age range 18-30 years, while the lowest belonged to age <40. This finding was in line with Khater et al. (2013), who detected the highest rate (33.3%) among those aged 21-30 years, (the highest percentage in those aged 20-29 years (Ghasemi et al., 2015), and disagreed with Khan et al. (2020) reported higher infection rate at age group 41-60 years (37.36%). In contrast, Nassef et al. (2018) and Mohamed (2020) noticed a non-significant relation to the age groups. The variation in young and adult human beings could be due to the difference in the immune status of persons, hygienic status, environmental conditions, different nutrition, the presence of sociocultural differences, and variations in terrain contribute to the exposure of varying age groups within the population to the infective form of the parasite.
The overall prevalence in cattle was in agreement with Tonouhewa et al. (2017), who found 12% (8%-17%) in Africa, Taalay et al., (2022), who recorded 12.2% in Pakistani cattle and Khattab et al., (2022), who reported 13.46% in Egyptian cattle. Majeed & Abbas (2018) found 14.91% in cattle in Basra City, Iraq, but disagree with Hassanain et al. (2013), who mentioned that the infection rate in Egyptian cattle was 29.4% using PCR. Shariatzadeh et al. (2021) recorded 16.94% in cattle and Fazel et al., (2021) recorded 56% in Iranian cattle. While Azizi et al. (2014) reported that the infection rate in Iranian cattle was 8.57%, Ji et al. (2023) detected that the infection rate in beef cattle was 2.9% in Korea. These differences could be due to the area of sample collection, number of collected samples, grassing type and distribution of stray cats in pastures, as well as the age of cattle (old age is more resistant to infection due to acquired immunity).
The highest percentage of T. gondii infection is seen in age group 2-4 years, while the lowest is at age <1-2 years. These results were in agreement with Blaga et al. (2019) and showed a strong age effect that adult cattle were more highly infected than calves in the age group (<8 months). Azizi et al. (2014) showed no significant correlation between age and infection rate in cattle. Kim & Seo (2023) showed the highest infection rate in the age group >6 years and the lowest prevalence among the age group <1 year. Sroka et al. (2020) reported that the infection rate of T. gondii increases with age of animals, indicating that age is a risk factor for T. gondii infection because older individuals are exposed to the parasite longer than younger individuals.
The infection rate was higher in male cattle (14.28%) than in females (8.10%). These results were in agreement with Fazel et al., (2021) and Kim & Seo (2023). They recorded that the prevalence of T. gondii was higher in male cattle than females. They disagreed with Sroka et al. (2020), who recorded the infection rate in female cattle higher than males. 

Conclusion
The detection of T. gondii in cattle blood explains that cattle meat is an important source of zoonotic transmission of toxoplasmosis to humans. We recommended establishing a strategy to control infection by health education on the zoonotic significance of toxoplasmosis, maintaining a high standard of personal hygiene, avoiding consuming raw or uncooked meat, and appropriate heat treatments for beef and milk.

Ethical Considerations

Compliance with ethical guidelines
All animal procedures were performed following the standards outlined in the guidelines of the Animal Welfare, Ethics and Experimentation Committee (No.: BMS/0231/016) of the Faculty of Veterinary Science, University of Babylon.

Funding
This research did not receive any grant from funding agencies in the public, commercial, or non-profit sectors. 

Acknowledgments
The author would like to thanks the butchers and farmers for their cooperation and assistance in facilitating the collection of blood samples from both themselves and their animals during the study.

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