Identification and genetic diversity of ticks in local sheep in Samarra city, northern of Baghdad, Iraq

This study reported hard tick (Ixodida: Ixodidae) infestation in sheep in Samara city, north of Baghdad, from June to December 2023. A total of 480 ticks were manually pulled out from the sheep with an ethanol-soaked tissue and preserved in plastic containers containing 70% ethyl alcohol. All samples were counted and examined individually under a light microscope to identify the genus. Preliminary microscopic examination revealed that the majority of the collected ticks were Hyalomma, with 91.4%, followed by Rhipicephalus (6.8%) and Ixodes (1.8%). Furthermore, Hyalomma was dominant across all months, with a peak relative abundance in October (100%). The peak relative abundances for Rhipicephlus (16%) and Ixodes (4.3%) occurred in July. Under further microscopic evaluation, morphological features demonstrated three species, namely, Hyalomma detritum (Koch), Hy. anatolicum (Koch) and Rhipicephalus sanguineus sensu lato (Latreille). To confirm genera and species, molecular diagnosis was performed, based on the COX1 gene, resulting in the identification of five species, namely, Hy. detritum , Hy. anatolicum , Hy. excavatum (Koch, 1844), Rh. sanguineus sensu lato and an unidentified Ixodes sp.  While the Ixodes scapularis isolation unexpectedly revealed genetic resemblance to North American samples, indicating a wider geographical spread, the Hyalomma and Rhipicephalus isolates shared ancestry with other Middle Eastern strains, demonstrating regional genetic stability. These results showed the comprehensive diversity of tick species in sheep, including Hyalomma, Rhipicephalus, and Ixodes, and also provided good epidemiological data to support the preventative procedures of tick-borne infections in Samarra city. 

Methods
Ethics. This project was approved by the ethical committee (ACUC) at the University of Baghdad (No.1667 on 22/03/2023).          

Study area. Samarra is a city in Salah Adin province, 120 km north of Baghdad. The city is bordered in the North-East by Diyala province, which shares borders with Iran (Figure 1). This study was conducted mainly in open farm areas in Samarra villages, where these areas are open to domestic and wild animals. Most of the farms lie at the border with Diyala province. The weather in this city is a typical desert climate, where it is extremely hot-dry in summer, and cold and dry to moderately wet in winter. The temperature ranges from 0°C on winter nights to over 50°C on summer days.

Parasite samples. Four hundred and eighty tick samples were obtained from more than 150 naturally infested, al-Awassi breed of sheep, from multiple farms in Samarra city (Figure 1). Sheep were thoroughly investigated for ticks during the period from June to December 2023. Hard ticks were collected from parts of the animals body which were devoid of wool, such as eyes, ears, udders, external genitalia and under tail areas. Samples were collected by a veterinarian twice per week in cooperation with the owners. After collection, hard ticks were transported to the parasitology laboratory of the College of Veterinary Medicine, University of Baghdad for microscopic examination then preserved in 10 ml of 70% ethyl alcohol for further processing.

Morphological identification of ticks. Ticks’ specimens were placed on a clean slide using double sided tape. Suitable forceps were used to manipulate ticks dorsally or ventrally and for spreading legs to examine internal structures. A small drop of glycerol was used to stabilize the tick on the slide. Tick samples were morphologically categorized under dissecting microscope into genera using taxonomic keys (Makwarela et al., 2024). Dorsal and ventral sides of adult ticks were precisely examined for distinguishing characteristics of structures under light microscopy. Subsequently, identified adults’ ticks were attached dorsally on the slides using the tape to photograph the specimens using a digital camera.

Extraction of DNA and PCR. Ticks were subjected to molecular analysis based on the mitochondrial cytochrome c oxidase subunit 1 gene (COX1). DNA extraction was performed on hard ticks, according to manufacturer’s guidelines using PCR Premix Kit (Bioneer, South Korea). Amplification of 680 bp of COX1 locus was done for molecular identification of tick species using one sets of primers. The primers were COX1-F 5’ATCATAAAKAYHTTGG 3’ andCOX1 -R 5’ GGGTGACCRAARAAHCA 3’ (Lv et al., 2014). PCR was done using a Veriti thermal cycler (ThermoFisher Scientific, USA) using the following parameters: initial denaturation step of 95 ◦C for 5 min, 30 cycles of denaturation at 95 ◦C for 45 s, annealing at 54–58 ◦C for 45 s and extension at 72 ◦C for 1 min, and a final extension of 72 ◦C for 5 min. All the PCR products were analysed by electrophoresis on agarose gel, stained with RedSafe (Intron, Korea), and visualized on a UV transilluminator. Amplicons were sent for sequencing (Macrogen, South Korea) and sequences were submitted to Basic Local Alignment Search Tool (BLAST) at the National Centre for Biotechnology information (NCBI). Multiple sequence alignment of the partial COX1 locus sequences were made by MUSCLE in SnapGene (Version 5.0.5. Iowa university, USA). A phylogenetic analysis was conducted using neighbor joining analyses with 500 bootstrap replicates in MEGA X (Kumar, et al., 2012).