This study was revised and approved by the Ethical Research Committee, Deanship of Scientific Research, King Khalid University, according to the ethical principles of human and animal research (Approval no. (ECM#2019-74) - (HAPO-06-B-001).
A cross-sectional study was conducted from May to October 2018 in four regions of Saudi Arabia: Riyadh (24°0′N, 45°30′E); Al-Kharj (24°08′N, 47°18E); Al-Hasa (25°23′N, 49°36E) and Al-Qassim (25°48′N, 42°52E) (Figure 1). All cows were apparently healthy at the time of sampling and were screened for tick infestation. Ticks found within 15 min were collected (2–5 ticks/infested animal) and placed in labelled tubes containing 70% ethanol that were individualised per cow. Ticks were identified to the species level by examination of their morphological features and use of morphological keys [15-17]. Each cow was categorised according to age and sex.
Blood samples were collected from each animal (2–5 ml) from the jugular vein into vacutainer tubes with ethylenediaminetetraacetic acid (EDTA) (BD Vacutainer® Tube, Gribbles Pathology, VIC, Australia) and transported in ice boxes to the molecular laboratory, Department of Biological Sciences, Faculty of Science and Humanities, Shaqra University, for DNA extraction.
Total genomic DNA (gDNA) was isolated from the blood samples using a Wizard® Genomic DNA Purification Kit (Promega, Madison, WI, USA) following the manufacturer’s instructions. Aliquots of between 50 and 100 µl of gDNA from each sample were stored at −20 °C prior to molecular analysis.
The primers were designed to target a conserved region that encoded ribosomal 18S rRNA (969 bp) and 238S rRNA (485 bp) to detect the DNA of Babesia  and Anaplasma , respectively (Table 1). Polymerase chain reaction (PCR) assays were performed in an automated BIO-RAD Thermal Cycler (BIO-RAD, Singapore) using one PCR master mix (GeneDireX, Taiwan). The PCR conditions for the Babesia amplification were as follows: incubation at 95 °C for 15 min; 35 cycles of 1 min at 95 °C; 30 s of annealing at 58 °C and 1 min at 72 °C; followed by a final extension for 5 min at 72 °C. Positive and negative controls were included. The PCR conditions used for Anaplasma amplification were as follows: an initial denaturation step at 95 °C for 15 min; then 40 cycles that consisted of 1 min of denaturation at 95 °C, 1 min of annealing at 55 °C, a 1 min extension at 72 °C, a final extension cycle at 72 °C for 7 min and cooling of the reaction mixtures at 15 °C. Distilled water samples were used as negative controls. Positive genomic controls of T. annulata and Anaplasma marginale were included. The amplifications were confirmed by electrophoresis on a 1.5% agarose gel, stained with Red Safe and examined by ultraviolet transillumination. A DNA molecular weight marker (100 bp DNA Ladder H3 RTU, GeneDireX, Taiwan) was used to estimate the size of the products. The PCR products of the positive samples were purified using a PCR Clean-Up & Gel Extraction Kit (GeneDireX, Taiwan) according to the manufacturer's instructions.
Sequencing and phylogenetic analyses
The purified PCR products were sequenced at Macrogene Lab Technology, Korea. The obtained sequences were assembled using ChromasPro software (ChromasPro 1.7, Technelysium Pty Ltd., Tewantin, Australia). The obtained sequences of Babesia and Anaplasma were submitted to GenBank and then compared with those available in the GenBank database by the US National Centre for Biotechnology Information Basic Local Alignment Search Tool (NCBI BLAST) (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Moreover, the sequences obtained from positive samples and sequences of validated species already available in GenBank were aligned using Bioedit software version 18.104.22.168 (Clustal W multiple alignment) . For taxonomic analyses, maximum-likelihood phylogenetic trees were constructed using Molecular Evolutionary Genetics Analysis (MEGA) software version X  with 500 bootstrap replications.
The significant differences regarding the prevalence of infections with Babesia and Anaplasma and their risk factors, such as sex and age of the cattle and their levels of tick infestation, were calculated by c2 tests using a program in Statistical Package for the Social Sciences (SPSS), version 20.0, at P< 0.05.