Molecular Identification of Toxoplasma gondii in the Native Slaughtered Cattle of Tehran Province, Iran

Background: Toxoplasmosis, caused by Toxoplasma gondii, is a common parasitic disease, affecting almost one-third of the world’s population. It is transmitted by ingestion of food or water contaminated with oocysts excreted by cats and the consumption of raw or undercooked meat from ruminants. This study aimed at molecular characterization of T. gondii in native cattle from West of Tehran, Iran. Methods: A total of 180 samples were collected from the cattle diaphragms (n=80) and heart muscles (n=100) from multiple slaughterhouses. The nested Polymerase Chain Reaction (PCR) assay was carried out to amplify the GRA6 gene of T. gondii. The PCR-Restriction Fragment Length Polymerase (PCR-RFLP) assay was also performed on positive samples, using Tru1I (MseI) restriction enzyme. Data were statistically analyzed using SPSS (v.15.0). Results: T. gondii was found in 38 out of 180 (21.1%) samples. The infection rate in heart muscle samples (16.66%) was significantly (p<0.05) higher than the diaphragm samples (4.44%). The PCR-RFLP pattern by MseI enzyme showed that 13 (7.22%) samples were genotype II, while 25 (13.88%) were genotype III, having statistically meaningful difference (p<0.05). No genotype I was found in the studied isolates. Conclusion: Based on our findings, the frequency of T. gondii was high in the study area. Therefore, educational programs need to be implemented in order to inform people about the risks of raw or undercooked meat consumption. © 2019, Shahid Sadoughi University of Medical Sciences. This is an open access article under the Creative Commons Attribution 4.0 International License.

mammals including humans Shariat Bahadori et al., 2019). Wild and domestic felids, as the final hosts, play a key role in the epidemiology of this zoonotic disease. In fact, felids are the only final hosts of T. gondii, which excrete oocysts through their feces (Dubey and Jones, 2008;Saadatnia and Golkar, 2012).
Toxoplasmosis is one of the most important problems not only in medicine, but also in veterinary fields (Dubey and Jones, 2008). The toxoplasmosis seroprevalence in the general population of Iran is estimated as 39.3% (95% CI, 33.0-45.7%; Daryani et al., 2014). Some evidences suggest that T. gondii imposes a considerable economic burden on livestock industries .
T. gondii may be found in a variety of animals, such as pigs, sheep, rabbits, goats, and domestic hen acting as intermediate hosts (Dubey et al., 2003;Mahami-Oskouei et al., 2017). In Iran, T. gondii infection has been detected in many domestic animals (e.g., cattle, sheep, and goats), which are used as food sources for people. T. gondii is transmitted to humans through exposure to water or food contaminated with cat-excreted oocysts. In addition, eating of raw or undercooked meat can also result in toxoplasmosis in human beings (Dubey and Jones, 2008).
Although, substantial research has been conducted on the transmission of infectious agents through consumption of foods of animal origin (e.g., meat) or ingestion of T. gondii oocysts, there are still some ambiguities. It should be noted that epidemiological studies still provide the most helpful strategy for detecting different sources of T. gondii infection in humans . Generally, different methods are used to identify T. gondii. Molecular methods, like Polymerase Chain Reaction (PCR), have many benefits, including quick identification, high sensitivity, and high specificity (MacPherson and Gajadhar, 1993). These methods can also distinguish between T. gondii alleles (Payne and Ellis, 1996). With this background in mind, the main purpose of this study was to genetically characterize T. gondii DNA in the native cattle of Tehran, Iran, via PCR-Restriction Fragment Length Polymerase (PCR-RFLP) assay.

Sample collection
This study was carried out in West of Tehran, Iran ( Figure 1). Samples were randomly collected from the diaphragms (n=80) and heart muscles (n=100) of 180 adult cattle from slaughterhouses of Qods, Shahriar, Malard, and Robat Karim counties in West of Tehran Province during March to December of 2017. The study protocol was approved by the Ethics Committee of Tarbiat Modares University, Tehran, Iran (ethics approval number: IR.MODARES.REC.1398.080). All samples were from the Holstein breed, and the majority of slaughtered cattle were local. All cattle were indigenous in the area and intended for human consumption. Sampling was carried out regardless of age and sex.

DNA extraction
First, 250 g of different segments of each muscle was collected and digested under sterile conditions, as described by Dubey (2016) with some modifications. To prepare the digestion solution (pepsin-hydrochloric acid digestion), 1 ml of distilled water was added to 10 ml of Hydrogen Chloride (HCl), 2.5 g of pepsin powder, and 5 g of Sodium Chloride (NaCl). Then, 50 g of each sample was mixed in 100 ml of digestion solution and placed in a warm bath for 30 min at 37 °C. Next, each digested muscles was passed through a two-layer-gauze. The solution was centrifuged for 15 min at 1500 rpm (252 x g), and the sediment was used for DNA extraction. For DNA extraction using a DNA purification kit (YTA Genomic DNA Extraction Kit, Favorgen, Taiwan), 25 g of each sediment solution was transferred to a sterile 1.5 ml microtube, following the manufacturer's instructions. The extracts were stored at -20 °C for molecular analysis.
In the nested-PCR assay, the primary PCR product (1 µl) was used for amplification of the internal sequences of GRA6 gene. The positive control contained T. gondii RH strain DNA, while the negative control was the nuclease-free water. The first PCR round was set up under the following conditions: one cycle of initial denaturation for five min at 94 °C, followed by 35 cycles of 94 °C for 30 s , 57 °C for 30 s , and 72 °C for 45 s . The final extension was done at 72 °C for 10 min. The nested-PCR condition was similar to the first reaction, whereas the annealing temperature in the second round was 56 °C. The amplified PCR products (5 μl) were analyzed on 2% agarose gel.

Genotyping of positive samples by PCR-RFLP assay
The Tru1I (MseI) restriction enzyme (Thermo Fisher Scientific, USA) was used in the PCR-RFLP assay on the positive samples (Abdoli et al., 2017). Digestion was performed in the reaction mixture at a final volume of 16 μl, as described by the manufacturer (5 μl of PCR product, 1 μl of 10X Buffer R, 9 μl of nuclease-free water, and 1 unit of MseI endonuclease) and incubated for 4 h at 65 °C. Then, the digested fragments were analyzed with 2% agarose gel. To better characterization, GRA6 sequences of T. gondii from RH type I, ME49 type II, and NED type III were collected from the GenBank (NCBI) and digested virtually by MseI restriction enzyme using NEBcutter (http://nc2.neb.com/NEBcutter2/) as shown in Figure 2.

GRA6 sequencing and phylogenetic analysis
In order to confirm the results of RFLP assay, the secondary PCR product of one positive sample was selected for each genotype and sequenced (Bioneer Company; South Korea). The sequences were edited and aligned using ClustalW program (http://www.ebi.ac.uk/Tools/ msa/clustalw2/). The maximum likelihood method was applied to plot the phylogenetic tree using MEGA 7. Also, the sequences were submitted at GenBank, NCBI.

Statistical analysis
Data were analysed by the SPSS.15 version using X 2 test. Results were considered significant at the 95% level (p<0.05).

Results
T. gondii DNA was found in 38 out of 180 (21.1%) cattle samples, which showed the amplicons with the size of ~344 bp (Figure 3). The infection rates in the heart muscle and diaphragm samples were 30 out of 180 (16.66%) and 8 out of 180 (4.44%), respectively, showing significant difference (p<0.05). Figure 4 demonstrates the results of GRA6 analysis of T. gondii in slaughtered cattle samples. The PCR-RFLP pattern by MseI enzyme showed that 13 (7.22%) samples were genotype II, while 25 (13.88%) were genotype III, having statistically meaningful difference (p<0.05). No genotype I was found in the studied isolates.
The analysis of GRA6 gene sequences corroborated the results of RFLP assay. The amplified GRA6 gene of the two isolates were sequenced and submitted to the GenBank under the accession numbers of MK055338 and MK055339. The alignment of our sequences showed 100% homology to other reported sequences in the GenBank. Using a phylogenetic tree, the phylogenetic relation of T. gondii isolates was compared with the same target gene in the other strains of T. gondii available in the GenBank ( Figure 5).

Discussion
The present study was carried out to detect and genetically characterize T. gondii DNA in the native slaughtered cattle of Tehran Province, Iran. In our study, 38 out of 180 samples (21.1%) were positive based on the GRA6 gene detection, therefore the prevalence of T. gondii in these regions is relatively high. In Iran, various studies have been conducted on different human and animal groups regarding the prevalence of toxoplasmosis. The prevalence rate has been estimated at 39% in the general population , 50% in immunodeficiency patients , 41% in pregnant women (Foroutan-Rad et al., 2016a), and 33% in blood donors (Foroutan-Rad et al., 2016b). Also, the prevalence rates have been estimated at 34% in felids , 27% in goats, and 31% in sheep . The rate of toxoplasmosis in Iranian cattle was 18.1% (9.2-28.5%) during a 30-year period from 1983 to 2012 . The contamination level of T. gondii varies in different regions of Iran. In a study by Anvari et al. (2018), 16.0% of different muscles collected from the slaughtered cattle in Zahedan (South-East of Iran) were contaminated with T. gondii. In another study from Lorestan Province, Western Iran, the seroprevalence of T. gondii in cattle was reported to be 28.73% (Hashemi, 2014). The mentioned findings, in line with the present study, are indicative of the high prevalence of toxoplasmosis in cattle of Iran. The high spread of toxoplasmosis in cattle in some areas may be due to several factors, including weather conditions, lack of routine treatment for feline toxoplasmosis, and contact with contaminated felids and Toxoplasma oocysts . However, the prevalence of T. gondii infection in the cattle in some provinces of Iran was lower, compared to the current study. For example, in a study in Ahvaz, South-West of Iran, only 4% of cattle were infected to T. gondii (Rahdar et al., 2012).
In the present study, the infection rate in heart muscle samples (16.66%) was significantly higher than the diaphragm samples (4.44%). Such findings were previously reported in muscle samples of cattle slaughtered in Switzerland (Berger-Schoch et al., 2011) and Tunisia (Lahmar et al., 2015). Indeed, the heart is known to be one of the most vulnerable to Toxoplasma cyst formation.
Also, the prevalence of T. gondii varies in different countries. In this way, Amdouni et al. (2017) stated that the infection rate of T. gondii was 19.3% in neck muscle samples of slaughtered cattle in North-West Tunisia. Another survey by Lopes et al. (2013) in Portugal showed that prevalence of T. gondii infection was 7.5% in cattle. These variations in the prevalence of T. gondii might be attributed to the type of food sources, methods of detection, geographical location, and sample analysis methods (Azizi et al., 2014). Also, it was shown that there is a meaningful relationship between weather and toxoplasmosis prevalence. In hot and wet climates and lower altitudes, it is typically more prevalent compared to cold and dry districts. This is consistent with longer viability of T. gondii oocyst in warm and humid environments .
T. gondii is categorized into three major strain types (type I, type II, and type III) that differ together in their epidemiological patterns, pathogenicity, and virulence (Chaichan et al., 2017;Sharif et al., 2017;Sibley et al., 2009). There was no evidence of genotype I in the present study. Though, in a PCR-RFLP assay conducted in North-West of Iran, 26 out of 150 (17.33%) chicken, beef, and lamb samples were positive for T. gondii, which all the samples were identified as genotype I (Mahami-Oskouei et al., 2017). The T. gondii genotype II has low pathogenicity and therefore allows the animal to survive until slaughter (Weiss and Kim, 2000). In our study, genotype II was found in 13 (7.2%) samples. In this regard, Lopes et al. (2015) showed that 60% of the cattle were seropositive for T. gondii, and three strains of genotype II were found in the heart samples of cattle, with predominant of genotype II (Lopes et al., 2015). In line with the present study, some evidences showed that genotype III was the most dominant genotype of T. gondii (Howe and Sibley, 1995).
The results of the current survey confirmed the presence of T. gondii in animals. Therefore, the risk of toxoplasmosis transmission due to contaminated meat consumption still needs to be considered as a public health problem (Dubey et al., 2014). To determine risk factors for infection of T. gondii in pregnant women, various studies worldwide have shown the significance of undercooked meat consumption as a contributor to human toxicity. It was found that consumption of undercooked meat of cattle and sheep was an important risk factor for infection of French pregnant women (Baril et al., 1999). Generally, there is a significant relationship between the incidence of toxoplasmosis and exposure of cows with cats. Examination of the risk factors for toxoplasmosis in large ruminants indicated that the presence of cats around cattle is a major factor in cattle infection (Ahmad and Qayyum, 2014).

Conclusion
The results showed that the frequency of T. gondii is high in cattle muscles in Tehran Province of Iran, and cattle infection plays an important role in the transmission of T. gondii to humans. Therefore, educational pro-grams need to be implemented to inform people about the risks of raw/undercooked meat consumption in this area. Also, it is suggested that T. gondii infection be studied in felid feces to reach a better understanding of the epidemiological aspects of toxoplasmosis in Iran.

Author contributions
A.D.Gh. and A.D. conceived the study and designed the study protocol; A.D. was supervisor of this research; A.D.Gh. did the experimental work and drafted the manuscript. Both authors read and approved the final version of the manuscript.

Conflicts of interest
The authors declare no potential conflicts of interest with respect to the research, authorship, and/or publication of this paper.