MATERIALS AND METHODS

Ethical approval

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.

Study period and location

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].

Figure 1: Geographical map of the study areas located in Wasit Province, Iraq.

Study design

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 = _Z_2_P_exp (1-_P_exp) / _d_2

where:

1. n = required sample size;

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

3. Pexp = expected prevalence (0.50);

4. d = desired precision (0.075).

Sample collection

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.

Molecular detection of T. orientalis

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.

DNA sequencing and phylogenetic analysis

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 analysis

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].