Genotyping of Toxoplasma gondii using GRA6 gene-based RFLP-PCR in raw milk from four livestock species in Sistan and Baluchestan Province, Iran

Genotyping of Toxoplasma gondii using GRA6 gene-based RFLP-PCR in raw milk from four livestock species in Sistan and Baluchestan Province, Iran | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Genotyping of Toxoplasma gondii using GRA6 gene-based RFLP-PCR in raw milk from four livestock species in Sistan and Baluchestan Province, Iran Mohammad Mirzaei, Mohammad Mohebbi, Bagher Amirheidari, Maryam Noshadokht This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8747226/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 10 You are reading this latest preprint version Abstract Background Toxoplasmosis is a zoonotic disease caused by the obligate intracellular protozoan Toxoplasma gondii , which is one of the primary causes of abortion, congenital infections, and stillbirth in animals and humans. In this study, we investigate the genetic diversity of T. gondii using the GRA6 gene in raw milk samples collected from various livestock in Sistan and Baluchestan Province, Iran. Methods A total of 224 raw milk samples (64 from cows, sheep, and goats; 32 from camels) were aseptically collected from animals in different herds across the province in 2024. Genomic DNA was extracted, and the presence of T. gondii was identified using nested-PCR targeting the GRA6 gene. Positive samples were genotyped using PCR-Restriction Fragment Length Polymorphism (RFLP) with the MseI enzyme. Representative amplicons were sequenced for phylogenetic confirmation. Results The molecular prevalence of T. gondii DNA was 11 samples (4.9%). RFLP analysis and subsequent sequencing revealed that all positive isolates belonged to genotype I of T. gondii . Conclusion This study confirms the presence of T. gondii DNA, particularly the potentially pathogenic genotype I, in raw milk from ruminants, especially small ruminants, in the region. Public health measures should urgently emphasize the risks of raw milk consumption. Health sciences/Diseases Biological sciences/Genetics Biological sciences/Microbiology Biological sciences/Molecular biology Toxoplasma gondii raw milk zoonosis genotyping PCR-RFLP GRA6 gene Figures Figure 1 Figure 2 Figure 3 1. Introduction Toxoplasmosis is a zoonotic disease caused worldwide by the apicomplexan parasite Toxoplasma gondii [ 1 ]. Domestic cats and other felids serve as definitive hosts, shedding resistant oocysts into the environment through their feces. Warm-blooded animals, including all major livestock species and humans, are intermediate hosts in which tissue cysts form [ 2 ]. In livestock populations, high seroepidemiological rates are often observed in species such as pigs, sheep, and goats. The transmission routes of T. gondii are multifaceted. Vertical or congenital transmission can occur when the parasite crosses the placenta and infects the fetus. Horizontal transmission routes include ingestion of resistant oocysts shed by felids in the environment, consumption of raw or undercooked meat containing tissue cysts, and consumption of unpasteurized milk harboring tachyzoites [ 3 ]. The resilience of T. gondii is notable, as tachyzoites can retain infectivity in various biological fluids for extended periods; for example, they survive 24 hours in saline, approximately 3 days in 3.5% serum albumin, 3 to 4 days in colostrum, and remarkably, 17 to 43 days in serum [ 4 – 5 ]. Direct detection of tachyzoites in the milk of ruminants such as sheep, goats, and cows has been documented, confirming milk as a potential transmission vehicle [ 6 – 11 ]. Despite these risks, raw milk is consumed by a significant portion of the global population. Advocates of raw milk consumption cite its superior nutritional quality, enhanced flavor, growing preference for natural and unprocessed food options, and the principle of individual choice as compelling reasons for favoring raw milk. There is a widespread belief that milk consumption benefits overall health. Conversely, raw milk has historically been recognized as an important source of pathogens capable of causing various human illnesses [ 12 ]. The economic impact of toxoplasmosis is primarily notable in livestock production due to its role in causing abortions and neonatal mortality in sheep and goats, leading to significant financial losses for producers. Additionally, through potential contamination of animal products, it poses a threat to public health [ 3 , 13 ]. Human infections with T. gondii are usually asymptomatic, although a percentage of individuals may experience clinical symptoms such as lymphadenopathy or ocular disease. The consequences of this disease are particularly severe during pregnancy, as infection can lead to spontaneous abortion, stillbirth, or congenital defects in the newborn. Infected infants who survive may face long-term consequences, including progressive cognitive impairments, hydrocephalus, microcephaly, chorioretinitis, or neonatal death [ 3 , 14 , 15 ]. In immunocompromised individuals, toxoplasmosis can reactivate and progress from an asymptomatic state to a disseminated, life-threatening condition. Clinical manifestations may include encephalitis, meningoencephalitis, or brain mass lesions. Without timely diagnosis and appropriate treatment, the outcome is often fatal [ 16 ]. Genetic diversity in T. gondii is characterized by three predominant clonal lineages (types I, II, and III), along with various atypical, recombinant, and hybrid strains. Type I strains are generally associated with high virulence, while types II and III typically exhibit reduced virulence in experimental models [ 17 ]. The GRA6 gene is a single-copy, polymorphic locus widely used for reliable differentiation of major types through PCR-RFLP, offering a distinct advantage over multicopy genes like B1, which are unsuitable for genotyping [ 18 ]. Several studies in Iran have reported the presence of T. gondii in raw milk. Specifically, a study in Sistan and Baluchestan Province using the B1 gene provided initial evidence of milk contamination [mo]. However, the B1 gene does not enable strain differentiation. Consequently, data on circulating T. gondii genotypes in dairy products from this ecologically distinct and agriculturally important region remain scarce. This study was designed to fill the knowledge gap by determining the predominant T. gondii genotypes in raw cow, sheep, goat, and camel milk in Sistan and Baluchestan Province using GRA6-based PCR-RFLP, thereby providing essential data for molecular epidemiology and more accurate risk assessment. 2. Materials and Methods 2.1. Sample Collection A total of 224 raw milk samples were randomly collected from healthy animals during routine milking in various herds in Sistan and Baluchestan Province in 2024. The sample set included 64 samples each from cows, sheep, and goats, and 32 samples from camels. Sampling followed standard hygiene protocols: teats and udders were cleaned and disinfected with 70% ethanol, and a portion of the initial milk was discarded before collection. Milk samples were then collected with sterile gloves into sterile containers. Data on location, herd management system (extensive/semi-intensive), and sampling date were recorded. Samples were immediately placed on ice and transported to the laboratory within 2–4 hours. 2.2. DNA Extraction Genomic DNA was extracted from each milk sample using a commercial DNA extraction kit (PEX, Pishgam, Iran) according to the manufacturer’s instructions. DNA samples were stored at − 20°C until polymerase chain reaction (PCR) was performed. 2.3. GRA6 Gene Amplification by Nested-PCR The primers used in this study were obtained from Pishgam, Iran. Primer sequences and references are listed in Table 1 . The first-round PCR was performed in a 25 µL reaction mixture containing 12.5 µL of 2X PCR Master Mix (Pishgam, Iran), 1.5 µL of each external primer (10 pmol/µL), 2 µL of DNA template, and 7.5 µL of nuclease-free water. Cycling conditions were: initial denaturation at 94°C for 5 minutes; 35 cycles of 94°C for 30 seconds, 54°C for 60 seconds, 72°C for 90 seconds; and final extension at 72°C for 7 minutes. Table 1 Primers used for amplifying the GRA6 gene of T. gondii by Nested-PCR. Amplicon size Reference Primer sequence Primer name - Lecordier et al, 1995 5'-GGCAAACAAAACGAAGTG-3' GRA6 F1 Lecordier et al, 1995 5'-CGACTACAAGACATAGAGTG-3' GRA6 R1 791 Fazaeli et al, 2000 5'-GTAGCGTGCTTGTTGGCGAC-3' GRA6 F2 Fazaeli et al, 2000 5'-TACAAGACATAGAGTGCCCC-3' GRA6 R2 Table 2 Prevalence of T. gondii DNA in raw milk samples from different animal species in Sistan and Baluchestan Province. Positive No. milk samples Species 1(1.6%) 64 Bovine 6(9.4%) 64 Ovine 4(6.3%) 64 Caprine 0 32 Camel 11(4.9%) 224 Total The obtained products were diluted 1:10 in sterile distilled water. The second stage was performed with the second primer pair, using 1 µL of the diluted product as template DNA, and the second cycle was conducted under the same temperature conditions, with only the annealing temperature adjusted to 60°C. The polymerase chain reaction (PCR) products were electrophoresed on a 2% agarose gel, and the results were recorded. DNA from the RH strain of T. gondii and nuclease-free water were used as positive and negative controls, respectively. 2.4. Genotyping by PCR-RFLP and Sequencing All Nested-PCR-positive samples were genotyped using RFLP. Ten microliters of the PCR product, 1.5 units of MseI enzyme, and 2 units of buffer R were incubated at 65°C for 4 hours. Electrophoresis was performed on an agarose gel, with the cleavage sites for types III, II, and I corresponding to 168 bp and 712 bp, 71 bp and 694 bp, and 71 bp, 168 bp, and 712 bp, respectively. For further phylogenetic analysis, the Nested-PCR amplicons were purified and sequenced by Pioneer Company (Iran). A molecular phylogenetic tree was then constructed based on the obtained sequences. For confirmatory phylogenetic analysis, GRA6 amplicons from three randomly selected positive samples (one from each host species) were purified and commercially sequenced by Pioneer Company (Iran). The obtained sequences were edited using Chromas software and aligned with reference sequences from GenBank using ClustalW in MEGA 12. A phylogenetic tree was constructed using the Neighbor-Joining method, with bootstrap analysis based on 1,000 replicates. 2.5. Statistical Analysis Data analysis was performed using SPSS software (version 27). The prevalence of T. gondii among different animal species was compared using the chi-square test or Fisher's exact test, as appropriate. A p-value of less than 0.05 was considered statistically significant. 2.6. Ethical statement This study involved the collection of raw milk samples from farm animals. As the research did not involve any experimentation or direct manipulation of animals, and samples were collected non-invasively during routine milking, obtaining a separate ethics code for this type of study was not mandatory according to our institution's guidelines. Nevertheless, the study protocol was conducted in accordance with the ethical guidelines of our institution. Verbal consent was obtained from the farm owners for sample collection." 3. Results 3.1. Prevalence of T. gondii in Raw Milk As shown in Figure 1, Nested-PCR successfully amplified the 791-bp fragment of the GRA6 gene from T. gondii DNA in milk samples. The overall prevalence of T. gondii DNA was 4.9% (11/224). The highest prevalence was observed in sheep milk (9.4%, 6/64), followed by goat milk (6.3%, 4/64) and cow milk (1.6%, 1/64). None of the 32 camel milk samples tested positive. The difference in prevalence among the four animal species was not statistically significant (p = 0.104) (Table 2). 3.2. Genotyping and Phylogenetic Analysis RFLP analysis of all 11 positive samples with MseI revealed identical digestion patterns, which are specific to type I strains of T. gondii . Sequencing of the GRA6 gene from three representative isolates (GenBank accession numbers: PX648581, PX648582, PX648583) confirmed the RFLP results. BLAST analysis showed 99-100% identity with reference type I strains. The phylogenetic tree structure placed all three sequenced isolates from this study in a clade with strong support alongside reference type I strains, clearly separating them from clades containing type II, type III, and atypical strains. 4. Discussion The present study is the first report on genotyping T. gondii in raw milk from Sistan and Baluchestan Province, Iran. While a previous study in this province identified the parasite using the B1 gene [6], our work advances understanding by identifying the circulating genotype, which is critical for epidemiological mapping and risk characterization. The overall molecular prevalence of 4.9% found here is generally comparable to the rate reported in the previous provincial study [6]. Minor variations may be attributed to the inherent sensitivity/specificity characteristics of different genetic targets (GRA6 vs. B1) [30]. The consistently higher prevalence in small ruminants (sheep and goats) in this study aligns with similar studies [9, 20-23], likely due to their broader grazing habits, which increase exposure to oocyst-contaminated environments [24]. The most significant finding was the exclusive identification of type I genotype in all positive milk samples. This result is consistent with several reports from Iran.Based on molecular genotyping of the GRA6 gene sequences of T. gondii in aborted sheep fetuses in southwestern Iran and Qazvin province, type I was identified [25]. These findings suggest that T. gondii type I infection could be a potential risk factor for abortion in pregnant ewes [26]. In brain samples of aborted sheep fetuses in Razavi Khorasan province, using PCR-RFLP based on the GRA6 locus, all isolated strains were of type I [27]. Additionally, analysis of T. gondii strains from Urmia using AluI restriction digestion revealed identical banding patterns in all three positive samples, confirming infection with genotype I in sheep [20]. Phylogenetic evaluation of T. gondii oocysts isolated from soil samples in Gilan province showed that the majority of sequences were closely related to type I strains of the parasite [17]. In a study conducted on livestock in northern Italy, type I was also identified as the dominant genotype [28]. In contrast, other studies from Iran and many parts of Europe and North America report type II as the most common lineage in animals and humans. Genotyping of T. gondii from aborted fetuses in another study revealed a distribution of 30% type I, 20% type II, and 50% atypical strains [29]. Analysis in sheep using PCR-RFLP identified type II and type III strains [30]. Molecular identification and genetic diversity of T. gondii in various tissues of sheep and goats in eastern Iran showed that among all positive samples, three genotypes and one mixed genotype were present using the MseI enzyme [31]. Results in Gilan province, based on PCR-RFLP analysis targeting the GRA6 gene, identified type II T. gondii in 30 brain samples of aborted sheep fetuses [32]. This discrepancy highlights significant geographical diversity in the distribution of T. gondii strains. The predominance of type I in this region raises potential public health concerns, as this genotype is associated with high virulence in mouse models. The complete genotypic homogeneity suggests a probable common source or a dominant local transmission cycle involving specific definitive and intermediate host interactions [23]. Given that milk and dairy products are staple foods, especially for vulnerable groups such as children, pregnant women, and immunocompromised individuals, the consumption of unpasteurized milk poses a significant public health risk in the context of zoonotic toxoplasmosis transmission. Primary infection can lead to severe fetal damage, including microcephaly, hydrocephalus, chorioretinitis, and even abortion. The detection of parasite DNA does not definitively prove the presence of live and infectious tachyzoites, though it indicates a certain contamination risk. The sample size for camels was relatively small, limiting the power to draw conclusions about their susceptibility or role in the epidemiology of toxoplasmosis in the region. Additionally, the absence of serological data from source animals prevents correlating milk shedding with the animal's immune status or stage of infection. 5. Conclusion This study confirms the contamination of raw sheep, goat, and cow milk in Sistan and Baluchestan province with T. gondii DNA and establishes type I genotype as the dominant strain in these products. While a previous study had confirmed its presence, our genotyping data provide a deeper layer of epidemiological insight crucial for risk assessment. These findings emphasize that raw milk, particularly from small ruminants, poses a potential zoonotic risk in the region. The detection of a genotype often associated with higher virulence underscores the importance of public health preventive measures. Given that cats are the definitive hosts of this protozoan and are present in many livestock units, controlling contact between cats and livestock, as well as their feed, is a fundamental priority for infection prevention. It is recommended that samples from aborted sheep fetuses in the province be used to facilitate broader investigations. Finally, to prevent human infections, training environmental health personnel, maintaining hygiene standards in livestock farming, and standardizing meat preparation and storage are essential. Declarations Acknowledgment No specific acknowledgments are applicable for this work Authors' Contribution Study design: M.M, B.A.H, M.N Writing – review & editing: M.M, M.M, Investigation: M.M, M.M, Validation: M.M, Conceptualization: M.M, M.M, Writing – original draf: M.M, Experimental design: M.M, M.M, B.A.H, M.N Conflict of Interest The authors have declared no conflicts of interest. Ethics We hereby declare all ethical standards have been respected in preparation of the submitted article. Funding this research did not receive any specific grant. Data Availability The data that support the findings of this study are available on request from the corresponding author. The nucleotide sequences of the Toxoplasma gondii GRA6 gene generated during this study have been deposited in the GenBank repository and will be automatically published under the accession numbers PX648581, PX648582, and PX648583 after final processing and validation by GenBank staff. References Nosrati MC, Ghasemi E, Shams M, Shamsinia S, Yousefi A, Nourmohammadi H, et al. Toxoplasma gondii ROP38 protein: Bioinformatics analysis for vaccine design improvement against toxoplasmosis. Microbial pathogenesis. 2020;149:104488. https://doi.org/10.1016/j.micpath.2020.104488 Robert-Gangneux F, Dardé M-L. Epidemiology of and diagnostic strategies for toxoplasmosis. Clinical microbiology reviews. 2012;25(2):264-96. https://doi.org/10.1128/cmr.05013-11 Dubey JP. Toxoplasmosis–a waterborne zoonosis. Veterinary parasitology. 2004;126(1-2):57-72. https://doi.org/10.1016/j.vetpar.2004.09.005 Sacks JJ, Roberto RR, Brooks NF. Toxoplasmosis infection associated with raw goat's milk. Jama. 1982;248(14):1728-32. https://doi.org/10.1001/jama.1982.03330140038029 Raisanen S, Saari K. The importance of trophozoites in transmission of toxoplasmosis: survival and pathogenity of Toxoplasma gondii trophozoites in liquid media. 1978. https://doi.org/10.1016/0306-9877(78)90072-5 Mohebbi, M., Mirzaei, M., Amir Heidari, B., & Noshadokht, M. (2025). Identification of Toxoplasma gondii B1 Gene in Raw Milk from Sistan and Baluchestan Provinces Using Nested-PCR. Archives of Razi Institute (Articles in Press). https://doi.org/10.22092/ari.2025.369740.3692 Dubey J, Verma S, Ferreira L, Oliveira S, Cassinelli A, Ying Y, et al. Detection and survival of Toxoplasma gondii in milk and cheese from experimentally infected goats. Journal of food protection. 2014;77(10):1747-53. https://doi.org/10.4315/0362-028X.JFP-14-167 Mancianti F, Nardoni S, Papini R, Mugnaini L, Martini M, Altomonte I, et al. Detection and genotyping of Toxoplasma gondii DNA in the blood and milk of naturally infected donkeys (Equus asinus). Parasites & Vectors. 2014;7(1):165. https://doi.org/10.1186/1756-3305-7-165 Dehkordi FS, Haghighi Borujeni MR, Rahimi E, Abdizadeh R. Detection of Toxoplasma gondii in raw caprine, ovine, buffalo, bovine, and camel milk using cell cultivation, cat bioassay, capture ELISA, and PCR methods in Iran. Foodborne Pathogens and Disease. 2013;10(2):120-5. https://doi.org/10.1089/fpd.2012.1311 Çulbasan V, Gungor C, Gundog DA, Koskeroglu K, Onmaz NE. Molecular surveillance of Toxoplasma gondii in raw milk and Artisan cheese of sheep, goat, cow and water buffalo origin. International Journal of Dairy Technology. 2023;76(4):948-54. https://doi.org/10.1111/1471-0307.12988 Khan S, Rafiq K, Khabir M, Khan M, Khan S, Khattak A, et al. Toxoplasma gondii in lactating animals: potential risk to milk consuming population in Khyber Pakhtunkhwa. Brazilian Journal of Biology. 2023;83:e267369. https://doi.org/10.1590/1519-6984.267369 Staal SJ, Kaguongo W. The Ugandan dairy sub-sector: targeting development opportunities. 2003. https://scholar.google.com/scholar_lookup?&title=The%20Ugandan%20dairy%20subsector%20targeting%20development%20opportunities&publication_year=2003&author=Staal%2CSJ&author=Kaguongo%2CWN Buxton D, Maley SW, Wright SE, Rodger S, Bartley P, Innes EA. Toxoplasma gondii and ovine toxoplasmosis: new aspects of an old story. Veterinary parasitology. 2007;149(1-2):25-8. https://doi.org/10.1016/j.vetpar.2007.07.003 Joynson D, Guy E. Laboratory diagnosis of toxoplasma infection. Toxoplasmosis: a comprehensive clinical guide Cambridge University Press, Cambridge, United Kingdom. 2001:296-318. https://scholar.google.com/scholar_lookup?title=Laboratory+diagnosis+of+Toxoplasma+infection&author=DHM+Joynson&author=EC+Guy&publication_year=2001&pages=296-318&doi=10.1017%2FCBO9780511527005.014 Higa LT, Araújo SM, Tsuneto L, Castilho-Pelloso M, Garcia JL, Santana RG, et al. A prospective study of Toxoplasma-positive pregnant women in southern Brazil: a health alert. Transactions of the Royal Society of Tropical Medicine and Hygiene. 2010;104(6):400-5. https://doi.org/10.1016/j.trstmh.2010.01.006 Schürmann D, Bergmann F, Albrecht H, Padberg J, Wünsche T, Grünewald T, et al. Effectiveness of twice-weekly pyrimethamine-sulfadoxine as primary prophylaxis of Pneumocystis carinii pneumonia and toxoplasmic encephalitis in patients with advanced HIV infection. European Journal of Clinical Microbiology and Infectious Diseases. 2002;21(5):353-61. https://doi.org/10.1007/s10096-002-0723-3 Hassani Lafmejan Pour H, Tavalla M, Valian HK, Mohebali M, Hafshejani SH, Latifi A, et al. Molecular detection of toxoplasma gondii oocytes in soil samples from guilan province, northern Iran. Iranian Journal of Public Health. 2024;53(3):654. https://doi.org/10.18502/ijph.v53i3.15147 Fazaeli A, Carter P, Darde M, Pennington T. Molecular typing of Toxoplasma gondii strains by GRA6 gene sequence analysis. International Journal for Parasitology. 2000;30(5):637-42. https://doi.org/10.1016/S0020-7519(00)00036-9 Lecordier et al, 1995 Tavassoli M, Esmaeilnejad B, Malekifard F, Soleimanzadeh A, Dilmaghani M. Detection of Toxoplasma gondii DNA in Sheep and Goat Milk in Northwest of Iran by PCR-RFLP. Jundishapur Journal of Microbiology. 2013;6(10). https://doi.org/10.5812/jjm.8201 Amairia S, Rouatbi M, Rjeibi MR, Nouasri H, Sassi L, Mhadhbi M, et al. Molecular prevalence of Toxoplasma gondii DNA in goats’ milk and seroprevalence in Northwest Tunisia. Veterinary Medicine and Science. 2016;2(3):154-60. https://doi.org/10.1002/vms3.29 Meftahi B, Abdollahian E, Nematollahi A, Razmi G. A Study of Association of Toxoplasma gondii Infection with Schizophrenia in Mashhad Area, Khorasan Razavi Province, Iran. Epidemiology and Health System Journal. 2021;8(2):88-92. https://doi.org/10.34172/ijer.2021.16 Alipour Amroabadi M, Rahimi E, Shakerian A. Study of the Seasonal and Geographical Prevalence of Toxoplasma gondii in milk of ruminants by nested-PCR. Food Hygiene. 2020;41:43-51. https://doi.org/10.30495/JFH.2020.583591.1206 Tenter, A. M., Heckeroth, A. R., & Weiss, L. M. (2000). Toxoplasma gondii: from animals to humans. International journal for parasitology , 30 (12-13), 1217-1258. https://doi.org/10.1016/S0020-7519(00)00124-7 Arefkhah N, Sarkari B, Asgari Q, Moshfe A, Khalafi MH, Mohammadpour I. Molecular genotyping of Toxoplasma gondii in sheep aborted fetuses reveals predominance of type I infection in southwest of Iran. Iranian Journal of Parasitology. 2020;15(3):374. https://doi.org/10.18502/ijpa.v15i3.4202 Habibi G, Imani A, Gholami M, Hablolvarid M, Behroozikhah A, Lotfi M, et al. Detection and identification of Toxoplasma gondii type one infection in sheep aborted fetuses in Qazvin Province of Iran. Iranian Journal of Parasitology. 2012;7(3):64. https://pubmed.ncbi.nlm.nih.gov/23109964/ Danehchin L, Razmi G, Naghibi A. Isolation and genotyping of Toxoplasma gondii strains in ovine aborted fetuses in Khorasan Razavi Province, Iran. The Korean journal of parasitology. 2016;54(1):15. https://doi.org/10.3347/kjp.2016.54.1.15 Battisti, E., Zanet, S., Trisciuoglio, A., Bruno, S., & Ferroglio, E. (2018). Circulating genotypes of Toxoplasma gondii in Northwestern Italy. Veterinary Parasitology , 253 , 43-47. https://doi.org/10.1016/j.vetpar.2018.02.023 Nourmohammadi M, Hamidinejat H, Tabandeh M, Goraninejad S, Bahrami S. Genotyping of Zoonotic Toxoplasm gondii Isolated from Aborted Fetuses of Ewes of Lorestan Province Based on SAG2، SAG3 and GRA6 Molecular Markers. J Ardabil Univ Med Sci 2017; 17 (3) :343-352. http://jarums.arums.ac.ir/article-1-1455-fa.html Zia-Ali N, Fazaeli A, Khoramizadeh M, Ajzenberg D, Dardé M, Keshavarz-Valian H. Isolation and molecular characterization of Toxoplasma gondii strains from different hosts in Iran. Parasitology research. 2007;101(1):111-5. https://doi.org/10.1007/s00436-007-0461-7 Kareshk TA, Mahmoudvand H, Keyhani A, Oliaee TR, Mohammadi M, Babaei Z, et al. Molecular detection and genetic diversity of Toxoplasma gondii in different tissues of sheep and goat in Eastern Iran. Tropical Biomedicine. 2017;34:681-90. http://eprints.lums.ac.ir/id/eprint/1054 Nosrati, M. R. C., Shemshadi, B., Shayan, P., Ranjbar, S., & Bahadori, A. E. (2020). High prevalence of Toxoplasma gondii infection in ovine aborted fetuses in Gilan Province, Iran: Molecular detection and genotype characterization. J Basic Res Med Sci , 7 , 53-62. https://jbrms.medilam.ac.ir/browse.php?sid=1&a_id=559&slc_lang=en&ftxt=1 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 21 Apr, 2026 Reviews received at journal 18 Apr, 2026 Reviews received at journal 16 Apr, 2026 Reviewers agreed at journal 27 Mar, 2026 Reviewers agreed at journal 27 Mar, 2026 Reviewers invited by journal 16 Mar, 2026 Editor assigned by journal 16 Feb, 2026 Editor invited by journal 12 Feb, 2026 Submission checks completed at journal 11 Feb, 2026 First submitted to journal 07 Feb, 2026 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. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-8747226","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":606949115,"identity":"1a624e2c-c102-4a51-867e-61185316453d","order_by":0,"name":"Mohammad Mirzaei","email":"","orcid":"","institution":"Shahid Bahonar University of Kerman","correspondingAuthor":false,"prefix":"","firstName":"Mohammad","middleName":"","lastName":"Mirzaei","suffix":""},{"id":606949116,"identity":"8988006f-1a77-4ccc-b12e-4f5d76d0f2c3","order_by":1,"name":"Mohammad Mohebbi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzElEQVRIiWNgGAWjYFACHgZmxgYGHn4QO6GABC0ykg0gLQYkaLExOADiEKNFt7334OPCHYd5jM+vTvzwwIBBnl/sAH4tZmfOJRvPPHOYx+zG280SQIcZzpydQEDLjRwzad42kJazG0BaEgxuE9Zi/hukxXjG2c0/iNVixgzSYsDfu41IW86cMZaeeSadR+IG7zaLBAMJIvxyvMfwc+EOa3v+/rObb/6osJHnlyagBQqaGRgkwColiFIOAnUMDPwHiFY9CkbBKBgFIwwAAHkYRgr9S+6lAAAAAElFTkSuQmCC","orcid":"","institution":"Shahid Bahonar University of Kerman","correspondingAuthor":true,"prefix":"","firstName":"Mohammad","middleName":"","lastName":"Mohebbi","suffix":""},{"id":606949117,"identity":"fce13f24-75a7-4ab1-9d1a-f02a62a2b15a","order_by":2,"name":"Bagher Amirheidari","email":"","orcid":"","institution":"Kerman University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Bagher","middleName":"","lastName":"Amirheidari","suffix":""},{"id":606949118,"identity":"deb2035e-6273-4775-9225-636e0e693fa1","order_by":3,"name":"Maryam Noshadokht","email":"","orcid":"","institution":"Kerman University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Maryam","middleName":"","lastName":"Noshadokht","suffix":""}],"badges":[],"createdAt":"2026-01-31 06:53:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8747226/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8747226/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":104876929,"identity":"504afb94-62bd-4d2d-aedb-3ad0d1596902","added_by":"auto","created_at":"2026-03-18 08:44:11","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":236570,"visible":true,"origin":"","legend":"\u003cp\u003eResults obtained from electrophoresis of Nested-PCR products for amplification of the GRA6 gene of \u003cem\u003eT. gondii\u003c/em\u003e. M: marker; –: negative control; +: positive control; G1, G2, G3: positive samples(791bp).\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8747226/v1/3690a79a2d706c1f42babe56.jpg"},{"id":104876837,"identity":"ad745715-79e4-42f9-8824-5bba0c0e2f35","added_by":"auto","created_at":"2026-03-18 08:43:52","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":108773,"visible":true,"origin":"","legend":"\u003cp\u003eResults using the MseI enzyme: M: marker; +: positive control; –: negative control; S: sheep, G: goat, positive samples (168/712 b p).\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-8747226/v1/785f4e7c132800f3fc184a8d.png"},{"id":104876915,"identity":"992a643a-a81f-40ac-83c8-c58b4b4b7e87","added_by":"auto","created_at":"2026-03-18 08:44:09","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":97112,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic analysis of isolates (T4, T5, T2) and reference strains; GTI KM372588.1 (Type I), ME 49 AF239285.1 (type II), Beverley AF239284.1 (type II), NED AF239286.1 (type III), RUB AF239290.1 (Atypical), FOU AF239288.1 (Atypical), and CASTELLE JX044209.1 (Atypical). The tree was constructed using the neighbor-joining method after bootstrapping with 1,000 repetitions.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8747226/v1/1899956690693bb799aa9b85.jpg"},{"id":104876967,"identity":"33de977a-e03c-4d9d-9741-ffc20aa49a2a","added_by":"auto","created_at":"2026-03-18 08:44:17","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":995972,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8747226/v1/fb6d669d-c25d-4e73-885e-ffaeadf9c6bf.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Genotyping of Toxoplasma gondii using GRA6 gene-based RFLP-PCR in raw milk from four livestock species in Sistan and Baluchestan Province, Iran","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eToxoplasmosis is a zoonotic disease caused worldwide by the apicomplexan parasite \u003cem\u003eToxoplasma gondii\u003c/em\u003e [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Domestic cats and other felids serve as definitive hosts, shedding resistant oocysts into the environment through their feces. Warm-blooded animals, including all major livestock species and humans, are intermediate hosts in which tissue cysts form [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn livestock populations, high seroepidemiological rates are often observed in species such as pigs, sheep, and goats. The transmission routes of \u003cem\u003eT. gondii\u003c/em\u003e are multifaceted. Vertical or congenital transmission can occur when the parasite crosses the placenta and infects the fetus. Horizontal transmission routes include ingestion of resistant oocysts shed by felids in the environment, consumption of raw or undercooked meat containing tissue cysts, and consumption of unpasteurized milk harboring tachyzoites [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The resilience of \u003cem\u003eT. gondii\u003c/em\u003e is notable, as tachyzoites can retain infectivity in various biological fluids for extended periods; for example, they survive 24 hours in saline, approximately 3 days in 3.5% serum albumin, 3 to 4 days in colostrum, and remarkably, 17 to 43 days in serum [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Direct detection of tachyzoites in the milk of ruminants such as sheep, goats, and cows has been documented, confirming milk as a potential transmission vehicle [\u003cspan additionalcitationids=\"CR7 CR8 CR9 CR10\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDespite these risks, raw milk is consumed by a significant portion of the global population. Advocates of raw milk consumption cite its superior nutritional quality, enhanced flavor, growing preference for natural and unprocessed food options, and the principle of individual choice as compelling reasons for favoring raw milk. There is a widespread belief that milk consumption benefits overall health. Conversely, raw milk has historically been recognized as an important source of pathogens capable of causing various human illnesses [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. The economic impact of toxoplasmosis is primarily notable in livestock production due to its role in causing abortions and neonatal mortality in sheep and goats, leading to significant financial losses for producers. Additionally, through potential contamination of animal products, it poses a threat to public health [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Human infections with \u003cem\u003eT. gondii\u003c/em\u003e are usually asymptomatic, although a percentage of individuals may experience clinical symptoms such as lymphadenopathy or ocular disease. The consequences of this disease are particularly severe during pregnancy, as infection can lead to spontaneous abortion, stillbirth, or congenital defects in the newborn. Infected infants who survive may face long-term consequences, including progressive cognitive impairments, hydrocephalus, microcephaly, chorioretinitis, or neonatal death [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. In immunocompromised individuals, toxoplasmosis can reactivate and progress from an asymptomatic state to a disseminated, life-threatening condition. Clinical manifestations may include encephalitis, meningoencephalitis, or brain mass lesions. Without timely diagnosis and appropriate treatment, the outcome is often fatal [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eGenetic diversity in \u003cem\u003eT. gondii\u003c/em\u003e is characterized by three predominant clonal lineages (types I, II, and III), along with various atypical, recombinant, and hybrid strains. Type I strains are generally associated with high virulence, while types II and III typically exhibit reduced virulence in experimental models [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The GRA6 gene is a single-copy, polymorphic locus widely used for reliable differentiation of major types through PCR-RFLP, offering a distinct advantage over multicopy genes like B1, which are unsuitable for genotyping [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSeveral studies in Iran have reported the presence of \u003cem\u003eT. gondii\u003c/em\u003e in raw milk. Specifically, a study in Sistan and Baluchestan Province using the B1 gene provided initial evidence of milk contamination [mo]. However, the B1 gene does not enable strain differentiation. Consequently, data on circulating \u003cem\u003eT. gondii\u003c/em\u003e genotypes in dairy products from this ecologically distinct and agriculturally important region remain scarce. This study was designed to fill the knowledge gap by determining the predominant \u003cem\u003eT. gondii\u003c/em\u003e genotypes in raw cow, sheep, goat, and camel milk in Sistan and Baluchestan Province using GRA6-based PCR-RFLP, thereby providing essential data for molecular epidemiology and more accurate risk assessment.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Sample Collection\u003c/h2\u003e \u003cp\u003eA total of 224 raw milk samples were randomly collected from healthy animals during routine milking in various herds in Sistan and Baluchestan Province in 2024. The sample set included 64 samples each from cows, sheep, and goats, and 32 samples from camels. Sampling followed standard hygiene protocols: teats and udders were cleaned and disinfected with 70% ethanol, and a portion of the initial milk was discarded before collection. Milk samples were then collected with sterile gloves into sterile containers. Data on location, herd management system (extensive/semi-intensive), and sampling date were recorded. Samples were immediately placed on ice and transported to the laboratory within 2\u0026ndash;4 hours.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. DNA Extraction\u003c/h2\u003e \u003cp\u003eGenomic DNA was extracted from each milk sample using a commercial DNA extraction kit (PEX, Pishgam, Iran) according to the manufacturer\u0026rsquo;s instructions. DNA samples were stored at \u0026minus;\u0026thinsp;20\u0026deg;C until polymerase chain reaction (PCR) was performed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. GRA6 Gene Amplification by Nested-PCR\u003c/h2\u003e \u003cp\u003eThe primers used in this study were obtained from Pishgam, Iran. Primer sequences and references are listed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The first-round PCR was performed in a 25 \u0026micro;L reaction mixture containing 12.5 \u0026micro;L of 2X PCR Master Mix (Pishgam, Iran), 1.5 \u0026micro;L of each external primer (10 pmol/\u0026micro;L), 2 \u0026micro;L of DNA template, and 7.5 \u0026micro;L of nuclease-free water. Cycling conditions were: initial denaturation at 94\u0026deg;C for 5 minutes; 35 cycles of 94\u0026deg;C for 30 seconds, 54\u0026deg;C for 60 seconds, 72\u0026deg;C for 90 seconds; and final extension at 72\u0026deg;C for 7 minutes.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePrimers used for amplifying the GRA6 gene of T. gondii by Nested-PCR.\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\u003eAmplicon size\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eReference\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePrimer sequence\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePrimer name\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLecordier et al, 1995\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5'-GGCAAACAAAACGAAGTG-3'\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGRA6 F1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLecordier et al, 1995\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5'-CGACTACAAGACATAGAGTG-3'\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGRA6 R1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cb\u003e791\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFazaeli et al, 2000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5'-GTAGCGTGCTTGTTGGCGAC-3'\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGRA6 F2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFazaeli et al, 2000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5'-TACAAGACATAGAGTGCCCC-3'\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGRA6 R2\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\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 DNA in raw milk samples from different animal species in Sistan and Baluchestan Province.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePositive\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNo. milk samples\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSpecies\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1(1.6%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBovine\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6(9.4%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eOvine\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4(6.3%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCaprine\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCamel\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e11(4.9%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e224\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTotal\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 obtained products were diluted 1:10 in sterile distilled water. The second stage was performed with the second primer pair, using 1 \u0026micro;L of the diluted product as template DNA, and the second cycle was conducted under the same temperature conditions, with only the annealing temperature adjusted to 60\u0026deg;C. The polymerase chain reaction (PCR) products were electrophoresed on a 2% agarose gel, and the results were recorded. DNA from the RH strain of \u003cem\u003eT. gondii\u003c/em\u003e and nuclease-free water were used as positive and negative controls, respectively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Genotyping by PCR-RFLP and Sequencing\u003c/h2\u003e \u003cp\u003eAll Nested-PCR-positive samples were genotyped using RFLP. Ten microliters of the PCR product, 1.5 units of MseI enzyme, and 2 units of buffer R were incubated at 65\u0026deg;C for 4 hours. Electrophoresis was performed on an agarose gel, with the cleavage sites for types III, II, and I corresponding to 168 bp and 712 bp, 71 bp and 694 bp, and 71 bp, 168 bp, and 712 bp, respectively. For further phylogenetic analysis, the Nested-PCR amplicons were purified and sequenced by Pioneer Company (Iran). A molecular phylogenetic tree was then constructed based on the obtained sequences.\u003c/p\u003e \u003cp\u003eFor confirmatory phylogenetic analysis, GRA6 amplicons from three randomly selected positive samples (one from each host species) were purified and commercially sequenced by Pioneer Company (Iran). The obtained sequences were edited using Chromas software and aligned with reference sequences from GenBank using ClustalW in MEGA 12. A phylogenetic tree was constructed using the Neighbor-Joining method, with bootstrap analysis based on 1,000 replicates.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Statistical Analysis\u003c/h2\u003e \u003cp\u003eData analysis was performed using SPSS software (version 27). The prevalence of \u003cem\u003eT. gondii\u003c/em\u003e among different animal species was compared using the chi-square test or Fisher's exact test, as appropriate. A p-value of less than 0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e\n\u003cp\u003e\u003cstrong\u003e2.6. Ethical statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study involved the collection of raw milk samples from farm animals. As the research did not involve any experimentation or direct manipulation of animals, and samples were collected non-invasively during routine milking, obtaining a separate ethics code for this type of study was not mandatory according to our institution\u0026apos;s guidelines. Nevertheless, the study protocol was conducted in accordance with the ethical guidelines of our institution. Verbal consent was obtained from the farm owners for sample collection.\u0026quot;\u003c/p\u003e"},{"header":"3. Results ","content":"\u003cp\u003e\u003cstrong\u003e3.1. Prevalence of \u003cem\u003eT. gondii\u003c/em\u003e in Raw Milk \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs shown in Figure 1, Nested-PCR successfully amplified the 791-bp fragment of the GRA6 gene from \u003cem\u003eT. gondii\u003c/em\u003e DNA in milk samples. The overall prevalence of \u003cem\u003eT. gondii\u003c/em\u003e DNA was 4.9% (11/224). The highest prevalence was observed in sheep milk (9.4%, 6/64), followed by goat milk (6.3%, 4/64) and cow milk (1.6%, 1/64). None of the 32 camel milk samples tested positive. The difference in prevalence among the four animal species was not statistically significant (p = 0.104) (Table 2). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2. Genotyping and Phylogenetic Analysis \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRFLP analysis of all 11 positive samples with MseI revealed identical digestion patterns, which are specific to type I strains of \u003cem\u003eT. gondii\u003c/em\u003e. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSequencing of the GRA6 gene from three representative isolates (GenBank accession numbers: PX648581, PX648582, PX648583) confirmed the RFLP results. BLAST analysis showed 99-100% identity with reference type I strains. The phylogenetic tree structure placed all three sequenced isolates from this study in a clade with strong support alongside reference type I strains, clearly separating them from clades containing type II, type III, and atypical strains. \u0026nbsp;\u003c/p\u003e"},{"header":"4. Discussion ","content":"\u003cp\u003eThe present study is the first report on genotyping \u003cem\u003eT. gondii\u003c/em\u003e in raw milk from Sistan and Baluchestan Province, Iran. While a previous study in this province identified the parasite using the B1 gene [6], our work advances understanding by identifying the circulating genotype, which is critical for epidemiological mapping and risk characterization. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe overall molecular prevalence of 4.9% found here is generally comparable to the rate reported in the previous provincial study [6]. Minor variations may be attributed to the inherent sensitivity/specificity characteristics of different genetic targets (GRA6 vs. B1) [30]. The consistently higher prevalence in small ruminants (sheep and goats) in this study aligns with similar studies [9, 20-23], likely due to their broader grazing habits, which increase exposure to oocyst-contaminated environments [24]. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe most significant finding was the exclusive identification of type I genotype in all positive milk samples. This result is consistent with several reports from Iran.Based on molecular genotyping of the GRA6 gene sequences of \u003cem\u003eT. gondii\u003c/em\u003e in aborted sheep fetuses in southwestern Iran and Qazvin province, type I was identified [25]. These findings suggest that \u003cem\u003eT. gondii\u003c/em\u003e type I infection could be a potential risk factor for abortion in pregnant ewes [26]. In brain samples of aborted sheep fetuses in Razavi Khorasan province, using PCR-RFLP based on the GRA6 locus, all isolated strains were of type I [27]. Additionally, analysis of \u003cem\u003eT. gondii\u003c/em\u003e strains from Urmia using AluI restriction digestion revealed identical banding patterns in all three positive samples, confirming infection with genotype I in sheep [20]. Phylogenetic evaluation of \u003cem\u003eT. gondii\u003c/em\u003e oocysts isolated from soil samples in Gilan province showed that the majority of sequences were closely related to type I strains of the parasite [17]. In a study conducted on livestock in northern Italy, type I was also identified as the dominant genotype [28].\u003c/p\u003e\n\u003cp\u003eIn contrast, other studies from Iran and many parts of Europe and North America report type II as the most common lineage in animals and humans. Genotyping of \u003cem\u003eT. gondii\u003c/em\u003e from aborted fetuses in another study revealed a distribution of 30% type I, 20% type II, and 50% atypical strains [29]. Analysis in sheep using PCR-RFLP identified type II and type III strains [30]. Molecular identification and genetic diversity of \u003cem\u003eT. gondii\u003c/em\u003e in various tissues of sheep and goats in eastern Iran showed that among all positive samples, three genotypes and one mixed genotype were present using the MseI enzyme [31]. Results in Gilan province, based on PCR-RFLP analysis targeting the GRA6 gene, identified type II T. gondii in 30 brain samples of aborted sheep fetuses [32]. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis discrepancy highlights significant geographical diversity in the distribution of \u003cem\u003eT. gondii\u003c/em\u003e strains. The predominance of type I in this region raises potential public health concerns, as this genotype is associated with high virulence in mouse models. The complete genotypic homogeneity suggests a probable common source or a dominant local transmission cycle involving specific definitive and intermediate host interactions [23]. Given that milk and dairy products are staple foods, especially for vulnerable groups such as children, pregnant women, and immunocompromised individuals, the consumption of unpasteurized milk poses a significant public health risk in the context of zoonotic toxoplasmosis transmission. Primary infection can lead to severe fetal damage, including microcephaly, hydrocephalus, chorioretinitis, and even abortion.\u003c/p\u003e\n\u003cp\u003eThe detection of parasite DNA does not definitively prove the presence of live and infectious tachyzoites, though it indicates a certain contamination risk. The sample size for camels was relatively small, limiting the power to draw conclusions about their susceptibility or role in the epidemiology of toxoplasmosis in the region. Additionally, the absence of serological data from source animals prevents correlating milk shedding with the animal\u0026apos;s immune status or stage of infection. \u0026nbsp;\u003c/p\u003e"},{"header":"5. Conclusion ","content":"\u003cp\u003eThis study confirms the contamination of raw sheep, goat, and cow milk in Sistan and Baluchestan province with \u003cem\u003eT. gondii\u003c/em\u003e DNA and establishes type I genotype as the dominant strain in these products. While a previous study had confirmed its presence, our genotyping data provide a deeper layer of epidemiological insight crucial for risk assessment. These findings emphasize that raw milk, particularly from small ruminants, poses a potential zoonotic risk in the region. The detection of a genotype often associated with higher virulence underscores the importance of public health preventive measures. Given that cats are the definitive hosts of this protozoan and are present in many livestock units, controlling contact between cats and livestock, as well as their feed, is a fundamental priority for infection prevention. It is recommended that samples from aborted sheep fetuses in the province be used to facilitate broader investigations. Finally, to prevent human infections, training environmental health personnel, maintaining hygiene standards in livestock farming, and standardizing meat preparation and storage are essential.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo specific acknowledgments are applicable for this work\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; Contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStudy design: M.M, B.A.H, M.N\u003c/p\u003e\n\u003cp\u003eWriting \u0026ndash; review \u0026amp; editing: M.M, M.M,\u003c/p\u003e\n\u003cp\u003eInvestigation: M.M, M.M,\u003c/p\u003e\n\u003cp\u003eValidation: M.M,\u003c/p\u003e\n\u003cp\u003eConceptualization: M.M, M.M,\u003c/p\u003e\n\u003cp\u003eWriting \u0026ndash; original draf: M.M,\u003c/p\u003e\n\u003cp\u003eExperimental design: M.M, M.M, B.A.H, M.N\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have declared no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe hereby declare all ethical standards have been respected in preparation of the submitted article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ethis research did not receive any specific grant.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study are available on request from the corresponding author.\u003c/p\u003e\n\u003cp\u003eThe nucleotide sequences of the \u003cem\u003eToxoplasma gondii\u003c/em\u003e GRA6 gene generated during this study have been deposited in the GenBank repository and will be automatically published under the accession numbers PX648581, PX648582, and PX648583 after final processing and validation by GenBank staff.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eNosrati MC, Ghasemi E, Shams M, Shamsinia S, Yousefi A, Nourmohammadi H, et al. \u003cem\u003eToxoplasma gondii\u003c/em\u003e ROP38 protein: Bioinformatics analysis for vaccine design improvement against toxoplasmosis. Microbial pathogenesis. 2020;149:104488. https://doi.org/10.1016/j.micpath.2020.104488\u003c/li\u003e\n\u003cli\u003eRobert-Gangneux F, Dard\u0026eacute; M-L. Epidemiology of and diagnostic strategies for toxoplasmosis. Clinical microbiology reviews. 2012;25(2):264-96. https://doi.org/10.1128/cmr.05013-11\u003c/li\u003e\n\u003cli\u003eDubey JP. Toxoplasmosis\u0026ndash;a waterborne zoonosis. Veterinary parasitology. 2004;126(1-2):57-72. https://doi.org/10.1016/j.vetpar.2004.09.005\u003c/li\u003e\n\u003cli\u003eSacks JJ, Roberto RR, Brooks NF. Toxoplasmosis infection associated with raw goat\u0026apos;s milk. Jama. 1982;248(14):1728-32. https://doi.org/10.1001/jama.1982.03330140038029\u003c/li\u003e\n\u003cli\u003eRaisanen S, Saari K. The importance of trophozoites in transmission of toxoplasmosis: survival and pathogenity of Toxoplasma gondii trophozoites in liquid media. 1978. https://doi.org/10.1016/0306-9877(78)90072-5\u003c/li\u003e\n\u003cli\u003eMohebbi, M., Mirzaei, M., Amir Heidari, B., \u0026amp; Noshadokht, M. (2025). Identification of \u003cem\u003eToxoplasma gondii\u003c/em\u003e B1 Gene in Raw Milk from Sistan and Baluchestan Provinces Using Nested-PCR. \u003cem\u003eArchives of Razi Institute \u003c/em\u003e(Articles in Press). https://doi.org/10.22092/ari.2025.369740.3692\u003c/li\u003e\n\u003cli\u003eDubey J, Verma S, Ferreira L, Oliveira S, Cassinelli A, Ying Y, et al. Detection and survival of \u003cem\u003eToxoplasma gondii\u003c/em\u003e in milk and cheese from experimentally infected goats. Journal of food protection. 2014;77(10):1747-53. https://doi.org/10.4315/0362-028X.JFP-14-167\u003c/li\u003e\n\u003cli\u003eMancianti F, Nardoni S, Papini R, Mugnaini L, Martini M, Altomonte I, et al. Detection and genotyping of \u003cem\u003eToxoplasma gondii\u003c/em\u003e DNA in the blood and milk of naturally infected donkeys (Equus asinus). Parasites \u0026amp; Vectors. 2014;7(1):165. https://doi.org/10.1186/1756-3305-7-165\u003c/li\u003e\n\u003cli\u003eDehkordi FS, Haghighi Borujeni MR, Rahimi E, Abdizadeh R. Detection of \u003cem\u003eToxoplasma gondii\u003c/em\u003e in raw caprine, ovine, buffalo, bovine, and camel milk using cell cultivation, cat bioassay, capture ELISA, and PCR methods in Iran. Foodborne Pathogens and Disease. 2013;10(2):120-5. https://doi.org/10.1089/fpd.2012.1311\u003c/li\u003e\n\u003cli\u003e\u0026Ccedil;ulbasan V, Gungor C, Gundog DA, Koskeroglu K, Onmaz NE. Molecular surveillance of \u003cem\u003eToxoplasma gondii\u003c/em\u003e in raw milk and Artisan cheese of sheep, goat, cow and water buffalo origin. International Journal of Dairy Technology. 2023;76(4):948-54. https://doi.org/10.1111/1471-0307.12988\u003c/li\u003e\n\u003cli\u003eKhan S, Rafiq K, Khabir M, Khan M, Khan S, Khattak A, et al. \u003cem\u003eToxoplasma gondii\u003c/em\u003e in lactating animals: potential risk to milk consuming population in Khyber Pakhtunkhwa. Brazilian Journal of Biology. 2023;83:e267369. https://doi.org/10.1590/1519-6984.267369\u003c/li\u003e\n\u003cli\u003eStaal SJ, Kaguongo W. The Ugandan dairy sub-sector: targeting development opportunities. 2003. https://scholar.google.com/scholar_lookup?\u0026amp;title=The%20Ugandan%20dairy%20subsector%20targeting%20development%20opportunities\u0026amp;publication_year=2003\u0026amp;author=Staal%2CSJ\u0026amp;author=Kaguongo%2CWN\u003c/li\u003e\n\u003cli\u003eBuxton D, Maley SW, Wright SE, Rodger S, Bartley P, Innes EA. \u003cem\u003eToxoplasma gondii\u003c/em\u003e and ovine toxoplasmosis: new aspects of an old story. Veterinary parasitology. 2007;149(1-2):25-8. https://doi.org/10.1016/j.vetpar.2007.07.003\u003c/li\u003e\n\u003cli\u003eJoynson D, Guy E. Laboratory diagnosis of toxoplasma infection. Toxoplasmosis: a comprehensive clinical guide Cambridge University Press, Cambridge, United Kingdom. 2001:296-318. https://scholar.google.com/scholar_lookup?title=Laboratory+diagnosis+of+Toxoplasma+infection\u0026amp;author=DHM+Joynson\u0026amp;author=EC+Guy\u0026amp;publication_year=2001\u0026amp;pages=296-318\u0026amp;doi=10.1017%2FCBO9780511527005.014\u003c/li\u003e\n\u003cli\u003eHiga LT, Ara\u0026uacute;jo SM, Tsuneto L, Castilho-Pelloso M, Garcia JL, Santana RG, et al. A prospective study of Toxoplasma-positive pregnant women in southern Brazil: a health alert. Transactions of the Royal Society of Tropical Medicine and Hygiene. 2010;104(6):400-5. https://doi.org/10.1016/j.trstmh.2010.01.006\u003c/li\u003e\n\u003cli\u003eSch\u0026uuml;rmann D, Bergmann F, Albrecht H, Padberg J, W\u0026uuml;nsche T, Gr\u0026uuml;newald T, et al. Effectiveness of twice-weekly pyrimethamine-sulfadoxine as primary prophylaxis of Pneumocystis carinii pneumonia and toxoplasmic encephalitis in patients with advanced HIV infection. European Journal of Clinical Microbiology and Infectious Diseases. 2002;21(5):353-61. https://doi.org/10.1007/s10096-002-0723-3\u003c/li\u003e\n\u003cli\u003eHassani Lafmejan Pour H, Tavalla M, Valian HK, Mohebali M, Hafshejani SH, Latifi A, et al. Molecular detection of \u003cem\u003etoxoplasma gondii \u003c/em\u003eoocytes in soil samples from guilan province, northern Iran. Iranian Journal of Public Health. 2024;53(3):654. \u003cu\u003ehttps://doi.org/10.18502/ijph.v53i3.15147\u003c/u\u003e\u003c/li\u003e\n\u003cli\u003eFazaeli A, Carter P, Darde M, Pennington T. Molecular typing of \u003cem\u003eToxoplasma gondii\u003c/em\u003e strains by GRA6 gene sequence analysis. International Journal for Parasitology. 2000;30(5):637-42. https://doi.org/10.1016/S0020-7519(00)00036-9\u003c/li\u003e\n\u003cli\u003eLecordier et al, 1995\u003c/li\u003e\n\u003cli\u003eTavassoli M, Esmaeilnejad B, Malekifard F, Soleimanzadeh A, Dilmaghani M. Detection of \u003cem\u003eToxoplasma gondii\u003c/em\u003e DNA in Sheep and Goat Milk in Northwest of Iran by PCR-RFLP. Jundishapur Journal of Microbiology. 2013;6(10). https://doi.org/10.5812/jjm.8201\u003c/li\u003e\n\u003cli\u003eAmairia S, Rouatbi M, Rjeibi MR, Nouasri H, Sassi L, Mhadhbi M, et al. Molecular prevalence of \u003cem\u003eToxoplasma gondii\u003c/em\u003e DNA in goats\u0026rsquo; milk and seroprevalence in Northwest Tunisia. Veterinary Medicine and Science. 2016;2(3):154-60. https://doi.org/10.1002/vms3.29\u003c/li\u003e\n\u003cli\u003eMeftahi B, Abdollahian E, Nematollahi A, Razmi G. A Study of Association of \u003cem\u003eToxoplasma gondii\u003c/em\u003e Infection with Schizophrenia in Mashhad Area, Khorasan Razavi Province, Iran. Epidemiology and Health System Journal. 2021;8(2):88-92. https://doi.org/10.34172/ijer.2021.16\u003c/li\u003e\n\u003cli\u003eAlipour Amroabadi M, Rahimi E, Shakerian A. Study of the Seasonal and Geographical Prevalence of \u003cem\u003eToxoplasma gondii\u003c/em\u003e in milk of ruminants by nested-PCR. Food Hygiene. 2020;41:43-51. https://doi.org/10.30495/JFH.2020.583591.1206\u003c/li\u003e\n\u003cli\u003eTenter, A. M., Heckeroth, A. R., \u0026amp; Weiss, L. M. (2000). Toxoplasma gondii: from animals to humans. \u003cem\u003eInternational journal for parasitology\u003c/em\u003e, \u003cem\u003e30\u003c/em\u003e(12-13), 1217-1258. https://doi.org/10.1016/S0020-7519(00)00124-7\u003c/li\u003e\n\u003cli\u003eArefkhah N, Sarkari B, Asgari Q, Moshfe A, Khalafi MH, Mohammadpour I. Molecular genotyping of \u003cem\u003eToxoplasma gondii\u003c/em\u003e in sheep aborted fetuses reveals predominance of type I infection in southwest of Iran. Iranian Journal of Parasitology. 2020;15(3):374. \u003cbr\u003e https://doi.org/10.18502/ijpa.v15i3.4202\u003c/li\u003e\n\u003cli\u003eHabibi G, Imani A, Gholami M, Hablolvarid M, Behroozikhah A, Lotfi M, et al. Detection and identification of \u003cem\u003eToxoplasma gondii\u003c/em\u003e type one infection in sheep aborted fetuses in Qazvin Province of Iran. Iranian Journal of Parasitology. 2012;7(3):64. https://pubmed.ncbi.nlm.nih.gov/23109964/\u003c/li\u003e\n\u003cli\u003eDanehchin L, Razmi G, Naghibi A. Isolation and genotyping of \u003cem\u003eToxoplasma gondii\u003c/em\u003e strains in ovine aborted fetuses in Khorasan Razavi Province, Iran. The Korean journal of parasitology. 2016;54(1):15.\u003cu\u003ehttps://doi.org/10.3347/kjp.2016.54.1.15\u003c/u\u003e\u003c/li\u003e\n\u003cli\u003eBattisti, E., Zanet, S., Trisciuoglio, A., Bruno, S., \u0026amp; Ferroglio, E. (2018). Circulating genotypes of Toxoplasma gondii in Northwestern Italy. \u003cem\u003eVeterinary Parasitology\u003c/em\u003e, \u003cem\u003e253\u003c/em\u003e, 43-47. https://doi.org/10.1016/j.vetpar.2018.02.023\u003c/li\u003e\n\u003cli\u003eNourmohammadi M, Hamidinejat H, Tabandeh M, Goraninejad S, Bahrami S. Genotyping of Zoonotic Toxoplasm gondii Isolated from Aborted Fetuses of Ewes of Lorestan Province Based on SAG2، SAG3 and GRA6 Molecular Markers. J Ardabil Univ Med Sci 2017; 17 (3) :343-352. http://jarums.arums.ac.ir/article-1-1455-fa.html\u003c/li\u003e\n\u003cli\u003eZia-Ali N, Fazaeli A, Khoramizadeh M, Ajzenberg D, Dard\u0026eacute; M, Keshavarz-Valian H. Isolation and molecular characterization of \u003cem\u003eToxoplasma gondii\u003c/em\u003e strains from different hosts in Iran. Parasitology research. 2007;101(1):111-5.\u003cu\u003e https://doi.org/10.1007/s00436-007-0461-7\u003c/u\u003e\u003c/li\u003e\n\u003cli\u003eKareshk TA, Mahmoudvand H, Keyhani A, Oliaee TR, Mohammadi M, Babaei Z, et al. Molecular detection and genetic diversity of \u003cem\u003eToxoplasma gondii\u003c/em\u003e in different tissues of sheep and goat in Eastern Iran. Tropical Biomedicine. 2017;34:681-90. http://eprints.lums.ac.ir/id/eprint/1054\u003c/li\u003e\n\u003cli\u003eNosrati, M. R. C., Shemshadi, B., Shayan, P., Ranjbar, S., \u0026amp; Bahadori, A. E. (2020). High prevalence of Toxoplasma gondii infection in ovine aborted fetuses in Gilan Province, Iran: Molecular detection and genotype characterization. \u003cem\u003eJ Basic Res Med Sci\u003c/em\u003e, \u003cem\u003e7\u003c/em\u003e, 53-62. https://jbrms.medilam.ac.ir/browse.php?sid=1\u0026amp;a_id=559\u0026amp;slc_lang=en\u0026amp;ftxt=1\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Toxoplasma gondii, raw milk, zoonosis, genotyping, PCR-RFLP, GRA6 gene","lastPublishedDoi":"10.21203/rs.3.rs-8747226/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8747226/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eToxoplasmosis is a zoonotic disease caused by the obligate intracellular protozoan \u003cem\u003eToxoplasma gondii\u003c/em\u003e, which is one of the primary causes of abortion, congenital infections, and stillbirth in animals and humans. In this study, we investigate the genetic diversity of \u003cem\u003eT. gondii\u003c/em\u003e using the GRA6 gene in raw milk samples collected from various livestock in Sistan and Baluchestan Province, Iran.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eA total of 224 raw milk samples (64 from cows, sheep, and goats; 32 from camels) were aseptically collected from animals in different herds across the province in 2024. Genomic DNA was extracted, and the presence of \u003cem\u003eT. gondii\u003c/em\u003e was identified using nested-PCR targeting the GRA6 gene. Positive samples were genotyped using PCR-Restriction Fragment Length Polymorphism (RFLP) with the MseI enzyme. Representative amplicons were sequenced for phylogenetic confirmation.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe molecular prevalence of \u003cem\u003eT. gondii\u003c/em\u003e DNA was 11 samples (4.9%). RFLP analysis and subsequent sequencing revealed that all positive isolates belonged to genotype I of \u003cem\u003eT. gondii\u003c/em\u003e.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eThis study confirms the presence of \u003cem\u003eT. gondii\u003c/em\u003e DNA, particularly the potentially pathogenic genotype I, in raw milk from ruminants, especially small ruminants, in the region. Public health measures should urgently emphasize the risks of raw milk consumption.\u003c/p\u003e","manuscriptTitle":"Genotyping of Toxoplasma gondii using GRA6 gene-based RFLP-PCR in raw milk from four livestock species in Sistan and Baluchestan Province, Iran","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-18 08:42:10","doi":"10.21203/rs.3.rs-8747226/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-04-21T04:27:03+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-18T14:02:55+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-16T17:13:19+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"117325767031141308576246269857207504782","date":"2026-03-27T20:06:16+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"69647706102997177590854533622657748640","date":"2026-03-27T11:32:11+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-03-16T13:19:57+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-02-16T13:49:36+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-02-12T20:35:34+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-02-12T02:24:25+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2026-02-08T04:45:19+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"39707178-b789-453b-a34a-e5cecf8495cf","owner":[],"postedDate":"March 18th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":64584516,"name":"Health sciences/Diseases"},{"id":64584517,"name":"Biological sciences/Genetics"},{"id":64584518,"name":"Biological sciences/Microbiology"},{"id":64584519,"name":"Biological sciences/Molecular biology"}],"tags":[],"updatedAt":"2026-05-05T05:23:50+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-18 08:42:10","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8747226","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8747226","identity":"rs-8747226","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}