Ethical approval
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).
Sampling procedures
The 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 was 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 morphological features and use of morphological keys [15, 16, 17]. Each cow was categorised according to age and gender.
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.
Molecular procedures
Total genomic DNA (gDNA) was isolated from the blood samples using the Wizard® Genomic DNA Purification Kit (Promega, Madison, WI, USA) following manufacturer’s instruction. Aliquots of between 50μl and 100µl 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 for ribosomal 18S rRNA (969 bp) and 238S rRNA(485 bp) to detect the DNA of Babesia [18] and Anaplasma [19] 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: one incubation step 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. PCR conditions of Anaplasma amplification were: an initial denaturation step at 95 °C for 15 min; then 40 cycles that consisted of 1 min denaturation at 95 °C, 1 min 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 reactions at 15 °C. Distilled water samples were used as negative controls. Positive genomic controls of Theileria annulata (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 (100bp DNA Ladder H3 RTU, GeneDireX, Taiwan) was used to estimate the size of the products. The PCR products of the positive samples were purified by use of 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). Morover, the sequences obtained from positive samples and to sequences of validated species already available in GenBank were aligned using Bioedit software version 7.0.5.3 (Clustal W multiple alignment) [20]. For taxonomic analyses, the maximum-likelihood phylogenetic trees were constructed using Molecular Evolutionary Genetics Analysis (MEGA) software version X [21] with 500 bootstrap replications.
Statistical analysis
The significant differences regarding the prevalence of infections with Babesia and Anaplasma and their risk factors, such as gender and age of the cattle and their levels of tick infestation, were calculated by x2 test using a Statistical Package for the Social Sciences (SPSS) program, version 20.0, at P< 0.05.