Description of microbial diversity associated with ticks Hyalomma dromedarii (Acari: Ixodidae) isolated from camels in Hail region (Saudi Arabia) using massive sequencing of 16S rDNA

Ticks are blood feeder able to transmit a wide diversity of microbes including pathogens. Therefore, it is of our interest to detect the diversity of microorganisms residing within ticks using massive sequencing of 16S rDNA. In this study, 200 adult ticks were collected from healthy camels in two localities from Hail province (Saudi Arabia). The analysis showed high microbial diversity dominated by the two domains (Archaea and Bacteria) associated with Hyalomma dromedarii from both regions. Proteobacteria (61.3%) and Firmicutes (31.2%) dominated the ticks from the Al Khotha region. While, the microbiome of ticks from the Al Gayed region was dominated by Proteobacteria (81.2%) and Firmicutes (9.2%). Twenty-three families were identified in the DNA-pool from the Al Gayed region, and was dominated by Pseudomonadaceae (45.37%), and Marinobacteraceae (14.39%) families. Francisellaceae (46%), Staphylococcaceae (24.26%) dominated the microbiome of the ticks collected from Al Gayed region. Thus, the genera Pseudomonas, Francisella, Proteus, Marinobacter, Glutamicibacter, Pedobacter, and Staphylococcus are largely distributed in the two identified microbiomes. This study concluded that ticks collected from the studied localities contained a wide range of microbial communities. These data have a great veterinary and medical importance in near future.

©Biomedical Informatics (2020) 603 microbiome of the ticks collected from Al Gayed region. Thus, the genera Pseudomonas, Francisella, Proteus, Marinobacter, Glutamicibacter, Pedobacter, and Staphylococcus are largely distributed in the two identified microbiomes. This study concluded that ticks collected from the studied localities contained a wide range of microbial communities. These data have a great veterinary and medical importance in near future.

Background:
Ticks are great resources for pathogens transmission, including viruses, Rickettsia, bacteria, and protozoa that can produce serious infections in human and animals worldwide [1]. Ticks and mesquites are the most common vectors for pathogens produce human and animal diseases, but ticks are considered the main vector of pathogens harming cattle worldwide [2]. A number of features of ticks enable them excellent vectors of pathogens, include the wide host range and tendency to feed on a number of hosts during life cycle ensure sufficient chance to obtain and pass on microorganisms, toughness and long life make them able to adapt and survive undesirable conditions, high frequency of host-vector contact, the high existing of ticks in both rural and urban areas and long attachment in the host which enable an ample time to transmit pathogenic agents.
There is a rising concern in the tick microorganism diversity, as it may induce significant effects of the transmission of pathogenic microbes, and the manipulation of tick microbial community may enhance the pathogens control [3]. Investigations on tick microbes demonstrated their medical and veterinary significance. The spread of tick vectors has led to the increase of tick-borne diseases and microbes associated with ticks [4]. Last decade, the uses of advanced techniques in molecular biology include next-generation sequencing (NGS) enable us to determine the microbial communities within ticks [3, 5, 6]. Common pathogens transmitted by ticks are rickettsia, borrelia and Babesia that cause severe infections to both human and animals. Borrelia burgdorferi sensu lato (B. afzelii, B. bavariensis, b gariniiand B. spielmanii. B) causes Lyme diseases (Lyme borreliosis), this bacterium belongs to the spirochaetes and helical-shaped bacteria. It is the main widespread tick-borne disease in US. B. burgdorferiis transmitted to human by blacklegged ticks [7,8]. It was found to be highly existed in the mild climate areas, and it was named after Lyme, a town in Connecticut, USA, where it was first emerged in 1975. Lyme disease is extensively spread, and has been documented in five continents and in more than 70 countries. Symptoms of this disease may include itchiness and redness of the skin, high fever, erythema migrans, neurological disorders and fatigued muscles [6,9,10].
Rickettsia is also a very popular pathogen transmitted by ticks; it is an obligate intracellular Gram-negative bacterium. This bacterium replicates usually in neutrophils and can spread diseases in human and animals [11][12][13]. The best example of diseases caused by Rickettsiaconorii is rickettsial spotted fever and also known as typhus which highly occurrences in many countries include USA, Asia, and Europe. This disease was first reported in 1936 in Russia. It has been reported that some people with chronic diseases, have underlying diseases [14]. Even though many studies have been conducted to identify microbial communities within ticks worldwide, still a little is known about the microbial diversities inhabiting ticks in Saudi, in particular Ha'il region. Therefore, it is of interest to detect the diversity of microorganisms residing within ticks using massive sequencing of 16S rDNA.

Methodology Study area and tick collection:
The study was conducted in some sites of Hail area. Hail city is located in the middle of the northern region of the Arabian Peninsula, between 25°-29° N and 38°-42° and has an average elevation of 900-1350 m above sea level. It is one of the major cities in the Kingdom of Saudi Arabia and considered as the fifth city in regard to its area. The local geology is dominated by the Arabian shield rock extending to steep wadies and hills. It is characterized by its limestone sand, which exists in the form of sand sheets and sand dunes. The tick samples (200 ticks: 4 to 5 specimen from each animal) were collected from healthy camels from locations of Al Gayed and Al Khotha regions of Hail province. One hundred alive adult ticks were collected from the two studied regions (20 healthy camels from each region). Tick samples were identified by using stereomicroscope and identified based on morphology [15,16]. Pooling was done for engorged ticks from the same camel while ticks were placed individually in 1.5 ml eppendorf tube. Ticks collected were stored at -20 °C until DNA extraction.

DNA extraction and quantification:
The extraction of DNA from tick samples was performed using a commercial DNA extraction kit with the Qiagen DNeasy Tissue kit (Qiagen) following the manufacturer's instructions with some modifications in the initial steps [17]. Briefly, after removal of ©Biomedical Informatics (2020) ethanol,the tick samples were placed in a sterile Petri dish and washed three times in sterile distilled water followed by one absolute ethanol bath, air dried and collected in sterile eppendorf tubes. Followed by washing with phosphate-buffered saline, 300 µl of 0.7M NaOH was added directly to pooled ticks and incubated at 56°C within hot air oven overnight. The ticks were crushed thoroughly using sterile motor and pestles and then the sample tubes were placed in a boiling water bath for 10 min. After cooling, 400 µl of sample solution withOTU disturbing Tick remnants, and placed within a sterile 2mL microcentrifuge tube. The proceeding steps are taken from The QIAamp DNA Mini Kit manufacturer's handbook, following the Tissue Protocol Incubate the samples at 70 °C for 10 minutes. The extracted DNA samples were pooled in to two groups based on location. DNA samples were quntified by macrogen. Inc (korea) using picogreen (Invitrogen, cat.#P7589 ) method using Victor 3 fluorometry.

Sample Preparation: 16S rRNA amplicon sequencing and data analysis:
The 16S RNA hypervariable regions were amplified by using universal primer Bakt-341F and Bakt-805R primers [18]. The genomic libraries for the Illumina sequencing were generated by ligating sequencing adapters and indices to purified PCR products using the Nextera XT Kit (Illumina, San Diego, CA, USA) according to the 16S rRNAmetataxonomics sequencing library preparation protocol (Illumina, San Diego, CA, USA). Followed by equimolar volume of induvidual libraries was pooled and sequenced on an Illumina's Miseq platform with pairedend 300 bp reads by MacrogenInc (Seoul, South Korea). The FLASH version 1.2.11 software was used to assemble MiSeq reads [19]. The taxonomy and alpha diversity were analysed by using QIIME software. The sequencing data is converted into raw data for the analysis.
Generation of raw data were done by Illumina sequencer generates raw images utilizing sequencing control software for system control and base calling through an integrated primary analysis software called RTA (Real Time Analysis). The BCL (base calls) binary is converted into FASTQ utilizing illumina package bcl2fastq. Table 1 shows the different phylum found in the two pools analyzed. The analyzed pools contained two main domains: Archaea (Euryarchaeota) and Bacteria (eight phylum: Actinobacteria, Bacteroidetes, Cyanobacteria, Deinococcus-Thermus, Firmicutes, Patescibacteria, Proteobacteria, and Verrucomicrobia). Only one domain (Bacteria) is characterizing the pool 1 with only four phylum (Actinobacteria, Bacteroidetes, Firmicutes, and Proteobacteria). Taxonomic profiling showed a high abundance of reads assigned to the phylum Proteobacteria in both pools analyzed. Firmicutes phylum is abundant in the first pool with more than 29540 OTU ID identified as compared to the second pool (3231 OTU ID). All these data are summarized in Table 1.

Quinella, Psychrobacter, Pedobacter, Stenotrophomonas, [Eubacterium]
ruminantium group, and Brevibacterium ( Figure 3B).  [36] conducted analysis for bacterial community in whole H. dromedarii ticks in Saudi Arabia via sequencing of the V3-V4 segment of 16S rRNA gene using Illumina MiSeq sequencer. These authors demonstrated that the most dominant bacterial types were Proteobacteria (98.12%) represented by the genus Francisellawith average abundance of 94.37% followed by Proteus (2.97%) and Acinetobacter (0.46%) and Pseudomonas (0.14%). The second bacterial type was Firmicutes (1.34%) represented by the genus Staphylococcus (0.51%), Salinicoccus (0.21%), Enterococcus (0.12%) and Solibacillus (0.1%). Actinobacteria (0.33%) was the third bacterial type represented essentially by Corynebacterium (0.25%) and the last one was the Bacteroidetes (0.16%). This study concluded that ticks associated with camels collected from Hail region (Saudi Arabia) had a wide diversity of microorganisms belonging mainly to the domains Archaea and Bacteria. Using the metagenomic approach, more than nine phylum, 53 families, and 94 genera were identified in the two DNA-pools studied. The obtained results highlighted the medical importance of ticks (H. dromedarii) as reservoir of pathogenic, opportunistic and symbiotic microorganisms. A program of veterinary surveillance must be programmed in order to identify known pathogenic microorganisms associated with H. dromedarii in Hail region.