The study protocol was reviewed and approved by the Scientific Committee, College of Veterinary Medicine, University of Wasit, Iraq, and the Institutional Animal Care and Use Committee (IACUC), University of Wasit (Approval No. WU.CVM.CVM-PD-1429; approved on June 25, 2025). All procedures involving animals were conducted in accordance with the institutional guidelines for the ethical care and use of animals in research and complied with internationally accepted principles for veterinary research involving animals. Blood and tick sampling were performed by licensed veterinarians using standard aseptic techniques and appropriate animal restraint to minimize pain, distress, and handling stress. Approximately 5 mL of jugular venous blood was collected using sterile, single-use needles into EDTA-anticoagulated tubes, while attached ticks were carefully removed using sterile forceps to avoid unnecessary tissue injury. All sampled animals were returned to their owners immediately after sample collection without any invasive intervention beyond routine veterinary procedures. Verbal informed consent was obtained from the owners of all participating cattle before sample collection. The study did not involve endangered or protected animal species, and no experimental infections, euthanasia, or procedures that caused prolonged discomfort were performed. Laboratory procedures involving potentially infectious materials were conducted in accordance with appropriate biosafety practices to protect personnel and prevent environmental contamination.

This cross-sectional study was conducted from July to August 2025 in Wasit Province, located in east-central Iraq, approximately 172 km southeast of Baghdad (Figure 1). The province extends between longitudes 44°40′ E and latitudes 32°00′ N and 33°50′ N and shares its eastern border with the Islamic Republic of Iran. The northeastern region is characterized by low-relief plains and anticlinal folds that receive drainage from adjacent mountainous areas. Wasit Province supports a substantial cattle population owing to favorable agroecological conditions, including access to transboundary marshlands, abundant water resources, and productive alluvial plains that facilitate livestock production [25–27].

A cross-sectional molecular epidemiological study was conducted involving 170 naturally tick-infested crossbred cattle selected from different localities of Wasit Province, Iraq. Animals were enrolled following clinical examination and confirmation of natural tick infestation. All cattle were managed under traditional grazing systems on natural pastures and were frequently exposed to ticks despite routine tick-control practices.

The required sample size was calculated according to the formula described by Thrusfield [28]:

n = Z²P_exp (1-P_exp) / d²

where:

1. n = required sample size;

2. Z = 1.96 (95% confidence interval [CI]);

3. P_exp = expected prevalence (0.50);

4. d = desired precision (0.075).

Under aseptic conditions, approximately 5 mL of jugular venous blood was collected from each animal into ethylenediaminetetraacetic acid (EDTA)-anticoagulated tubes. Hard ticks attached to different body regions of each animal were carefully removed using sterile forceps and placed into individually labeled sterile plastic containers. Blood and tick samples were transported to the laboratory under refrigerated conditions and stored at −20°C until molecular analysis. Information on the age and sex of each animal was recorded for subsequent risk-factor analysis.

Genomic DNA was extracted from both blood and tick samples using the gSYNC™ DNA Extraction Kit (Geneaid Biotech, New Taipei City, Taiwan) according to the manufacturer's instructions. DNA concentration and purity were evaluated using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).

Conventional PCR targeting the 18S rRNA gene was performed using a primer pair designed from the T. orientalis Dunya-30-IRAQ reference sequence (GenBank accession no. OQ779975.1) with Primer3Plus (Wageningen University, Wageningen, the Netherlands). The PCR mixture (20 μL) consisted of 5 μL DNA template, 1 μL forward primer, 1 μL reverse primer, and 13 μL nuclease-free water in AccuPower® PCR PreMix tubes (Bioneer, Daejeon, South Korea).

PCR amplification was carried out using a T100 Thermal Cycler (Bio-Rad Laboratories, Hercules, CA, USA) under the following cycling conditions: initial denaturation at 94°C for 7 min; 30 cycles of denaturation at 94°C for 1 min, annealing at 58°C for 30 s, and extension at 72°C for 1 min; followed by a final extension at 72°C for 7 min.

Amplified PCR products were separated by electrophoresis on 1.5% agarose gels stained with ethidium bromide and electrophoresed at 100 V and 80 mA for 90 min. Positive controls consisted of DNA obtained from recently confirmed Iraqi T. orientalis isolates [24], whereas nuclease-free water served as the negative control. Amplified products were visualized under an ultraviolet transilluminator, and the expected amplicon size was 505 bp.

PCR-positive DNA samples from both cattle and tick specimens, together with the corresponding primers, were submitted to Macrogen Inc. (Seoul, South Korea) for bidirectional DNA sequencing using the modified Sanger sequencing method. The obtained nucleotide sequences were edited and aligned using the ClustalW algorithm. Subsequently, representative sequences were submitted to the National Center for Biotechnology Information (NCBI) GenBank database. Phylogenetic relationships and sequence homology analyses were performed using Molecular Evolutionary Genetics Analysis version 11 (MEGA 11).

Statistical analyses were performed using GraphPad Prism version 8 (GraphPad Software Inc., San Diego, CA, USA). The Chi-square (χ²) test was used to evaluate differences among categorical variables. Statistical significance was defined as p < 0.05, with significance levels indicated as follows: p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), and p < 0.0001 (****).

The odds ratio (OR), relative risk (RR), number needed to treat (NNT), and 95% CI were calculated using MedCalc Statistical Software version 22 (MedCalc Software Ltd., Ostend, Belgium) [29].

Based on clinical examination, the incidence and risk of tick infestation varied significantly according to the age and sex of the study cattle (Table 1). The incidence of tick infestation differed significantly among age groups (p = 0.0149). Cattle aged >3–6 years exhibited the highest infestation rate (36.47%) and RR (0.9388; p = 0.0008), whereas cattle aged <1 year showed the lowest infestation rate (13.53%) and RR (0.2918). Intermediate infestation rates were observed in cattle aged 1–3 years (28.82%) and >6 years (21.18%).

Sex was also significantly associated with tick infestation. Female cattle exhibited a significantly higher infestation rate than males (84.71% vs. 15.29%; p = 0.0039). Likewise, the RR of tick infestation was markedly higher in females (6.3855; p = 0.0001) than in males (0.3335).

Conventional PCR detected T. orientalis DNA in 21 of 170 (12.35%) cattle blood samples and 16 of 170 (9.41%) corresponding tick samples (Figure 2).

The prevalence of T. orientalis infection differed significantly among age groups (p = 0.043) (Table 2). The highest prevalence was observed in cattle aged 1–3 years (14.29%) and >3–6 years (14.52%), whereas lower prevalence was observed in cattle aged <1 year (8.70%) and >6 years (8.33%).

Similarly, cattle aged >3–6 years exhibited the highest OR (1.5376) and RR (1.2676), whereas animals aged >6 years showed the lowest OR (0.5859) and RR (0.6496). These findings indicate that cattle aged >3 to 6 years had the greatest likelihood of T. orientalis infection.

According to sex, male cattle exhibited a significantly higher prevalence of T. orientalis infection than females (19.23% vs. 11.11%; p = 0.0445). Likewise, the OR (1.9048) and RR (1.6129) were significantly greater in males than females (p = 0.0001).

Sequencing analysis of the detected T. orientalis isolates resulted in the submission of nucleotide sequences to the NCBI GenBank database under the isolate designations IS.HA.C1–IS.HA.C21 for cattle isolates and IS.HA.T1–IS.HA.T16 for tick isolates. The corresponding accession numbers ranged from PX851761.1 to PX851781.1 for cattle isolates and from PX851782.1 to PX851797.1 for tick isolates.

Comparative sequence analysis showed nucleotide identities of 78.76% to 100% for cattle isolates and 98.16% to 100% for tick isolates, with sequence variation of 0.001% to 0.05%. Phylogenetic analyses performed separately for cattle and tick isolates revealed that all cattle-derived T. orientalis isolates clustered closely with the Turkish reference isolate HQ197736.1. In contrast, tick-derived isolates exhibited the highest sequence identity with one Chinese isolate (PQ207062.1) and two Turkish isolates (OR211412.1 and HQ197736.1), suggesting close genetic relationships between Iraqi isolates and geographically neighboring T. orientalis strains (Tables 3 and 4; Figures 3–6).

Information regarding T. orientalis infection in cattle remains limited in Iraq [24], although recent studies conducted in Asia [30, 31], Africa [32, 33], the Americas [34], and Europe [35, 36] have demonstrated that this tick-borne parasite is associated with considerable morbidity, production losses, and, in some cases, mortality in cattle. In the present study, the molecular prevalence of T. orientalis was 12.35% in cattle and 9.41% in their naturally infesting ticks. These findings provide the first simultaneous molecular evidence of T. orientalis infection in cattle and associated ticks from Wasit Province, Iraq.

Compared with previous reports, the prevalence observed in this study was higher than those reported from Ethiopia (2.2%) [37], Turkey (5.6%) [38], and Egypt (8.8%) [39], but was comparable with reports from Vietnam (13.8%) [40] and Pakistan (15.0%) [41]. Lower prevalence than the present study has also been reported in northeastern Thailand (30.1%) [42], Kazakhstan (33.3%) [43], China (14.27%–36.5%) [30, 44], Malaysia (49.76%) [45], Japan (10%–64.8%) [46], and India (83.3%) [31]. Such wide geographical variation in the prevalence of bovine theileriosis is likely attributable to differences in climatic conditions, tick vector abundance, cattle management systems, host immunity, diagnostic approaches, and the genetic diversity of circulating Theileria strains [47].

Environmental factors strongly influence tick ecology, with warm and humid climates generally favoring tick survival and transmission, thereby increasing disease prevalence [48]. In addition, farm management practices, including grazing systems, biosecurity measures, and nutritional status, may alter host susceptibility by increasing physiological stress [49]. Farms managed as small-scale or sideline enterprises often have limited resources for tick control, hygiene, and routine veterinary surveillance, which may further increase the risk of blood parasite transmission [50]. Moreover, genetic variation among Theileria species, including the emergence of strains with different levels of virulence or antimicrobial resistance, may further contribute to regional differences in disease epidemiology [51]. Variability in reported prevalence may also reflect differences in molecular diagnostic procedures, including pre-analytical sample handling, analytical protocols, and biological factors affecting parasite detection.

The present study demonstrated that T. orientalis infection was significantly associated with animal age. The highest prevalence was observed in cattle aged 1–3 years and >3–6 years, whereas the highest risk of infection was detected in cattle aged >3–6 years. Likewise, the prevalence of PCR-positive tick samples was greatest among cattle aged 1–3 years. Interpretation of age-related susceptibility should be approached with caution because previous studies have not consistently distinguished between epidemic and endemic disease conditions [6, 52–54].

Previous investigations have reported inconsistent findings regarding the influence of age on T. orientalis infection. In Australia, T. orientalis was detected microscopically in a 4-day-old calf and by quantitative PCR in both newborn and fetal calves [55, 56]. Similarly, Mekata et al. [57] reported that calves born to infected dams were PCR-negative during the first 30 days after birth, whereas 88% became infected by 4 months of age, suggesting that vertical transmission may require several months before becoming detectable under low tick challenge conditions.

In Malaysia, Ola-Fadunsin et al. [45] reported no significant difference in infection prevalence between cattle aged ≤1 year (44.5%) and 1–≤2 years (42.22%), whereas significantly higher prevalence was observed in animals aged 2–≤5 years (52.52%) and >5 years (59.64%). Likewise, Selim et al. [39] demonstrated that asymptomatic cattle older than 3 years had a significantly greater prevalence of T. orientalis infection (13.1%; p < 0.003) than cattle aged <1 year (1.6%) or 1–3 years (5.6%). Conversely, cattle residing in endemic regions, including Iraq, may gradually develop partial protective immunity following repeated exposure to infected ticks, resulting in persistent infection without overt clinical disease [14, 20, 58, 59]. Therefore, higher prevalence in older animals may reflect cumulative exposure rather than increased susceptibility to clinical theileriosis.

Male cattle exhibited significantly higher infection prevalence, OR, and RR than female cattle. In contrast, the prevalence of T. orientalis infection in ticks was not significantly associated with host sex, although ticks collected from male cattle showed a numerically higher risk of infection.

Previous studies have reported inconsistent associations between sex and T. orientalis infection. Selim et al. [39] found no significant difference between female (9.2%) and male (7.5%) cattle, whereas Ola-Fadunsin et al. [45] reported a higher prevalence among females (51.33%) than males (43.89%). The discrepancy between the present findings and previous reports may reflect differences in herd structure, management practices, and animal utilization. In the present study, most female cattle were maintained for dairy production, whereas male cattle were primarily reared for beef production under more extensive grazing conditions, which may increase their exposure to tick vectors.

Although T. orientalis has previously been reported in neighboring countries, including Turkey, Iran, and Pakistan, molecular phylogenetic information from Iraq has been lacking despite the country's strategic geographical position linking Asia and the Mediterranean region. In the present study, phylogenetic analysis demonstrated that all cattle-derived T. orientalis isolates clustered closely with the Turkish isolate HQ197736.1. In contrast, tick-derived isolates exhibited the greatest sequence identity with one Chinese isolate (PQ207062.1) and two Turkish isolates (OR211412.1 and HQ197736.1).

These findings suggest regional genetic connectivity and may reflect transboundary movement of infected animals or tick vectors. The greater genetic diversity observed among tick isolates may be explained by ticks' ability to acquire parasites from multiple infected hosts throughout their life cycle, allowing them to harbor a broader range of T. orientalis genotypes than those circulating within individual cattle populations. Recent outbreaks reported in Asia and Australia have been associated with highly pathogenic genotypes, including the Ikeda genotype, which has expanded into previously unaffected regions [6]. Consequently, the present findings contribute important baseline molecular evidence regarding the circulation and potential dissemination of T. orientalis lineages in Iraq. However, the statement regarding "haplotype diversity and network analysis showing shared versus unique haplotypes between cattle and ticks" should be removed because such analyses were not performed in this study.

The 18S rRNA gene remains one of the most reliable molecular markers for detecting T. orientalis because of its high copy number and the presence of conserved regions interspersed with hypervariable domains that enable accurate differentiation from other apicomplexan parasites [60–62]. Furthermore, the conserved nature of the 18S rRNA gene facilitates phylogenetic reconstruction and comparison of Theileria isolates worldwide, providing a robust framework for investigating evolutionary relationships and genotype diversity [63–66].

Nevertheless, several limitations should be acknowledged. The study was limited to a single province in Iraq, which may not fully represent the nationwide epidemiology of T. orientalis. In addition, phylogenetic characterization was based solely on the 18S rRNA gene. More discriminatory genetic markers, such as the MPSP gene, ITS regions, mitochondrial genes, or whole-genome sequencing, were not investigated. Consequently, genotype-specific characterization, identification of polymorphic sites, and differentiation of major genotypes, including Ikeda, Chitose, and Buffeli, were beyond the scope of the present study. Future nationwide molecular surveillance incorporating multiple genetic markers and larger sample sizes is therefore warranted to improve understanding of the genetic diversity, transmission dynamics, and epidemiology of T. orientalis in Iraq.