Molecular Identification of Ehrlichia canis in Rhipicephalus Sanguineus Ticks from Siirt Province


 This study was performed on Ehrlichia canis positive ticks collected from dogs to perform sequencing of their 16S rRNA genetic section using the PCR method. The collection of ticks was performed from a total of 60 dogs in the Siirt province, Turkey. A total of 250 ticks were collected and morphologically investigated. All ticks were identified as Rhipicephalus sanguineus sensu lato (s.l). Ehrlichial DNA was detected by the PCR method performed on 38 (15.2 %) of the ticks. The E. canis strains obtained as a result of the sequence analysis were found to be 100% identical to the American Texas (MH620194), Indian (KX766395), and Egyptian (MG564254) strains. This study thereby has identified a zoonotic agent from the R. sanguineus ticks collected from the dogs in the Siirt province.


INTRODUCTION
Ticks are responsible for the spread of various pathogens that infect both humans and animals, including various bacteria species, helminths, protozoa, and viruses [1,2]. Worldwide, Ehrlichia canis is the most important species of Ehrlichia in dogs, and its principal vector is the brown dog tick Rhipicephalus sanguineus sensu lato (s.l) [3][4][5]. E. canis is a gram-negative, obligate-intracellular bacteria belonging to the Rickettsiales order of the Anaplasmataceae superclass [6,7], and causes the potentially fatal Canine monocytic ehrlichiosis (CME) [4][5][6]8,9]. E. canis causes an infection that could potentially last for a lifetime in dogs by infecting their blood mononuclear cells [10,11]. Clinical symptoms of the disease include fever, depression, lack of appetite, weight loss, hemorrhage, epistaxis, gastrointestinal symptoms (vomitus, diarrhea, etc.), and respiratory disorders. The most frequent laboratory fi ndings consist of thrombocytopenia, leukopenia, anemia, and hypergammaglobulinemia [6,12]. The diagnosis of CME is performed by enzyme-linked immunosorbent assay (ELISA) and immunofl uorescence antibody assay (IFA) methods, the latter of which is considered as the golden standard [11,13]. In the diagnosis of the disease PCR methods (Polymerase chain reaction, real-time PCR) are used with its high sensitivity and specifi city [11,14]. The ticks of the Ixodidae family transmit the rickettsia pathogens like Ehrlichia and Anaplasma through the biological path [1]. Vectors of E. canis are the nymphs and adults of the Rhipicephalus sanguineus ticks, which are commonly known as the brown dog tick [4,[6][7][8]. Since the transovarial transmission of E. canis has not been demonstrated, the larvae are insignifi cant in terms of infection [10]. While they are primarily a dog parasite, these ticks can also infest other hosts, including humans [2]. Although these ticks are present worldwide, they are most frequently encountered in tropic and sub-tropic climates [6]. The aim of this study is to determine the prevalence of E.canis in ticks collected from dogs in Siirt province by PCR method. The 16S rRNA gene region of the positive samples were sequenced, and the phylogenetic tree analysis was performed, and results recorded in GenBank.

Sample selection
The collection of ticks was performed from a total of 60 dogs from the Siirt province, located in the Southeastern Anatolia Region of Turkey (37° 55' N, 41° 57' E). Siirt province is in a semi-arid climate region, where the average highest and lowest temperatures range between 36.9 o C and 18.9 o C in summer, and 8.7 o C and -0.5 o C in winter. Puddles are frequently present during the summer. Once the dogs were inspected, the ticks were collected into the individually labeled 25 ml collection cups that contained 70% ethyl alcohol and transported to the laboratory.

Morphological identifi cation of ticks and DNA extraction
The identifi cation of the ticks was performed using the methods suggested by Walker et al. (2000) and Estrada-Peña et al. (2004) [15,16]. Each sampled tick was washed with 70% ethyl alcohol before DNA extraction. The ticks were then placed into Eppendorf tubes individually, that were subjected to a freeze-thaw process and then crushed using a sterile glass rod. The Invitrogen PureLink™ Genomic DNA Mini Kit (USA, K182002) was used to isolate the DNA from the samples, as per the protocols suggested by the commercial company. The obtained DNA was stored at -20 °C until the PCR process.

PCR Amplifi cation
The Nested PCR method was used to identify E. canis DNA. In the initial phase, the primers ECC (5´ AGAACGAACGCTGGCGGCAAGC-3´) and ECB (5´ CGTATTACCGCGGCTGCTGGCA-3´) were used to amplify the 16S rRNA gene section, while in the second phase, the E. canis specifi c primer ECAN5 (5-CAATTATTTATAGCCTCTGGCTATAGGA-3´) and the primer HE3 (5´-TATAGGTACCGTCATTATCTTCCCTAT-3´) were used [17][18][19]. The protocol suggested in the studies of Ayan et al. (2019) and Ayan et al. (2020) was used for the PCR [1,20]. A 1.5% agarose gel was then prepared, and was stained using RedSafe™ Nucleic Acid Staining Solution. The PCR products were run through the agarose gel, and images were obtained using a gel imaging device (Syngene Bio Imaging System). Ten percent of the positive PCR products (3 samples) that were suitable for sequencing were sent to a commercial company (BM Labosis, Ankara, Turkey) for two-way sequence analysis.

Sequence and Phylogenetic Analys is
The purifi ed PCR products were subjected to a two-way sequence analysis using an Applied Biosystems 377 DNA Sequencer device. 16S rRNA sequences for the sequenced isolates were registered to the GenBank with access numbers MW405833.1, MW408001.1, MW408000.1. The sequences with registry numbers M73227.1, AY262124.1, NR 157649.1, NR 074500.2, AF069758.1, MG564254.1, KX766395.1, MH620194.1 obtained from the GenBank were sorted in the MEGA 7 software using the Clustal W algorithm. The phylogenetic tree was then reconstructed using the Neighbor-joining model and Bootstrap tests (1000 repeats). The evolutionary similarity between the isolates was determined using the Maximum Composite Likelihood test. An UPGMA dendrogram was then created using the evolutionary similarity between E. canis isolate sequences, MEGA 7 software, and the Neighbor-joining model.

Ethical approval
Ethical approval for this study was obtained from the Siirt University Local Ethics Committee for Animal Experiments (Decision number 2020/04-01).

RESULTS
A total of 250 ticks were collec ted and morphologically identifi ed. All ticks were identifi ed as R. sanguineus (s.l.). E.canis DNA was detected using t he Nested PCR method in 38 (15.2 %) of the ticks (389 bp) (Fig.1). Figure 2   high bootstrap similarity value of 100% for E. Canis. Anaplasma ovis was taken as the outer group (AY262124.1).

DISCUSSION
Transmitting numerous pathogens: viruses, bacteria, and parasites, ticks present a serious health hazard for both humans and animals [21]. Ixodidae ticks are vectors for various diseases that affect both humans and animals living in tropical and subtropical climates in particular [22]. Diseases transmitted by ticks, like ehrlichiosis, have increased in frequency all around the world, threatening both human and animal health [23]. While E. canis is encountered worldwide, its presence and incidence rate varies following the geographical distribution of its vectors [24].
Studies performed in different geographical regions of the world report varying rickettsia infection prevalence for ticks. Latrofa [26]. Other study covering Tunisia and Morocco reported 16.3% prevalence of E. canis in ticks and a prevalence of 2.9% for R. sanguineus ticks specifi cally [27]. In studies that investigated the E. canis presence in ticks in Israel, Iran, and Cameroon, revealed prevalence of 10.4% [28], 16.66% [29], and 21% [30] respectively.
Studies performed in Turkey reported E. canis prevalence in dogs to be between 0% and 69.4% [31,32]. Due to the limited number of studies on the presence of E. canis in Turkey, the actual distribution and prevalence of the infection is unknown [33]. A study performed on ticks from the Thrace region of Turkey (İstanbul, Edirne, Tekirdağ, and Kırklareli provinces) reported a prevalence of 21.25% on a tick pool from 127 specimens [23]. Ayan et al. (2019) performed a study in the province of Van, on the R. sanguineus ticks and reported a 20% prevalence of E. canis, while the study of Aktas et al. (2009) reported a prevalence of 1.23% in a study performed on the salivary glands of ticks [34].
Since the amplifi cation of the 16S rRNA gene is widely used to determine Ehrlichia DNA [11], the 16S rRNA region was targeted for the molecular identifi cation of Ehrlichia species in this study as well. As a result of the study, E. canis DNA was detected in 38 (15.2%) of 250 R. sanguineus ticks.
The results of the present study are similar to some other studies in the literature [23,26,28,29], while higher compared to other studies [11,25,34]. The difference between the study results might be due to the geographical locations, different climate, sample size, sampling period, tick species and stages, the presence of suitable reservoirs, and the study method.
While some studies have extracted DNA from tick pools [23,28] or salivary glands [34], the DNA in the present study were extracted from the whole ticks, in line with numerous other studies in the literature [1,11,29].
While it's primarily known as a pathogen for dogs, some studies report the presence of E. canis in domestic ruminants [11,34,35] and in different tick species (R. bursa, R. sanguineus, D. marginatus) of Ixodidae family [22]. The fact that in this study E. canis was observed in R. sanguineus ticks supports such previous studies [19,22,30].
The E. canis strains identifi ed through 16S rRNA gene sequence analysis amplifi ed from R. sanguineus tick species were found to be 100% similar to American Texas (MH620194), Indian (KX766395), and Egyptian (MG564254) strains.
The most prominent vector known for E. canis is the R. sanguineus tick, but in a world where climate change, vector expansion, and pathogen distribution occur beyond expected limits, vectorial zoonotic diseases might increase and the vectors for pathogens might change.

CONCLUSION
This study is the fi rst to report the molecular detection of E.canis in R. sanguineus ticks in dogs of the Siirt province. The study has identifi ed a zoonotic agent in the R. sanguineus ticks collected from dogs of the Siirt province, which might cause disease in humans [36]. The results indicate that authorized establishments should implement protection and control measures to protect humans from the disease.

Authors' contributions
BAÇ and ÖYÇ conceived of the study, and participated in its design and coordination and helped to draft the manuscript. AA, ÖOK, ABY and ÖOA carried out the lab work (Morphological identifi cation of Tick and DNA extraction, PCR Amplifi cation, Sequence and Phylogenetic Analysis). All authors read and approved the fi nal manuscript.

Declaration of confl icting interests
The author(s) declared no potential confl icts of interest with respect to the research, authorship, and/or publication of this article.

Statement of Informed Consent
The owner understood procedure and agrees that results related to investigation or treatment of their companion animals, could be published in Scientifi c Journal Acta Veterinaria-Beograd.