Molecular identification of Anaplasma / Ehrlichia spp based on 16S rRNA gene in Hyalomma ticks in border line of Iran-Pakistan CURRENT STATUS: POSTED

Background Anaplasmosis / Ehrlichiosis tick-borne diseases human subtropical regions of the world. Due to infection both animals and humans and monitoring of ticks and the pathogens they carry, an extensive survey was conducted in border line of Iran-Pakistan of Sistan and Baluchistan, southeast corner of Iran in 2016-2017, where animal husbandry is the main activity of people and every week thousands of cattle cross the borders into the countries. The aim of the survey was to determine the prevalence and geographical distribution of Anaplasmosis / Ehrlichiosis agents in Hyalomma spp hard ticks. Ticks were collected, identified and processed for Anaplasma / Ehrlichia spp DNA detection. Results Six Hyalomma species were found in the region, where Hy. anatolicum was the most prevalent species collected on goats, cattle, and camel. Anaplasma / Ehrlichia genomes were found in 68.3% of the specimens. Anaplasma ovis, A.marginale, and E.ewingii DNAs prevalence were 81.82, 9.09, and 9.09% in the infected ticks respectively. DNA sequence and phylogenetic analysis of the 16SrRNA gene confirmed the detection of these three anaplasmosis agents while they had 99-100% identity with the strains previously reported in genbank from different parts of the world.

but there is little research on the anaplasmosis/ehrlichiosis and Hyalomma spp and whether they are the probable vectors of Anaplasma /Ehrlichia spp is still unproven, thus necessitating this study. Our objective was to study the presence and diversity of Anaplasma / Ehrlichia spp in Hyalomma spp ticks isolated from different domestic animals in Sistan and Baluchestan province, which is one of the animal husbandry poles of the country.

Tick species and abundance
Hard tick specimens were collected in rural areas of Chabahar, Sarbaz and Sib & Suran districts situated in the southeast corner of Iran and were tested for presence of Anaplasma by screening nested PCR assays with specific primers against the 16S rRNA gene of the bacteria. A total of 1020 Hyalomma ticks were collected in the study areas. These ticks belonged to six species including Hy.  Table 1).
Hyalomma anatolicum was the most prevalent species in all three districts of the study area. The number of Hy. analiticum ticks were significantly (P<0.01) higher than other five species including the second prevalent species (Hy. dromedarii). There were no significant variations between the frequencies of Hy. marginatum, Hy. dromedarii, andHy. asiaticum (Fig. 1). Due to low number of Hy. detricum and Hy. schulzei, these two species were excluded from analysis.

Anaplasma / Ehrlichia infection in ticks
By use of broad-spectrum EHR primers, 256 out of 1020 collected ticks (25%) were tested for the presence of Anaplasma's or related species 16SrRNA gene. Result of PCR assays revealed presence of Anaplasma /Ehrlichia genomes in 68.3% (175 out of 256) of the selected specimens. The species, number and prevalence of Anaplasma /Ehrlichia in Hyalomma spp ticks at each collection site are shown in Table 2. The rate of Anaplasma spp /E.ewingii infections was similar (67.8-69.2%) in Hy. anatolicum, Hy. dromedarii, Hy. asiaticum, and Hy. marginatum specimens. This rate was higher in Hy. detricum (2 out of 2, 100%) and lower in Hy. schulzei (2 out of 5, 40%) than other four species. A subset of positive PCR specimens against Anaplasma /Ehrlichia genome in ticks were sequenced and the consensus sequences were deposited in Gen Bank. Details of the Anaplasma / Ehrlichia spp positive samples are listed in Table 3.

Sequence and phylogenetic analysis
Analysis of the sequence data showed that A.ovis was the most prevalent (18 out of 22, 81.82%) Anaplasma species in the study area. All of the strains of A.ovis isolated in this study were identical to each other and to the other Iranian strains and to the strains from China (Accession number: MG869525) and Russia (Accession number: KC484563). In addition of A. ovis, two A. marginale isolates (9.09%) and two E. ewingii isolates (9.09%) were found in the selected ticks. Sequences of A. marginale in this study were identical to their counterparts from USA, Tajikistan, and China. It was the same for the isolated strains of E. ewingii obtained in this study which were identical with the isolates from Australia, USA, Brazil, Thailand, Iran, China, and Uganda. The sequence similarities between the isolated strains of A. marginale and or E. ewingii with the available data in Genbank was more than 99% to 100%.
The phylogenetic analysis of Anaplasma /Erhrlichia species was performed using the sequences obtained in this study in combination with the available data retrieved from Genbank. The bacterial species were clustered in four different clades includingI) A. ovis, II) A. marginale, III) A. platys-A.
phagocytophilum-A. odocoilei, IV) A. centrale-A. capra (Fig. 2). Interestingly all E. ewingii isolates were associated with the branches of clade III. This analysis showed no clear geographical pattern or further association with host among the A. ovis, An. marginale, or E. ewingii isolates.

Discussion
This is the first comprehensive study of Hyalomma ticks attached to domestic animals and their associated Anaplasma /Ehrlichia species conducted in border line of Iran-Pakistan, southeast corner of Iran. The results show that there are six infesting Hyalomma spp ticks and that there are at least three anaplasmosis agent that can be transmitted through a tick bite. This result is generally in accordance with observations on hard ticks and pathogens from animals in other parts of the country [8,14,15], while there are some distinctive results in this study.
In this study, Hy. anatolicum-infested animals were found the most common and the widest geographical range. This species were reported as the most prevalent hard tick previously from most parts of the country including borderline of Iran-Iraq [16], centre [17], south-eastern and northwestern [15,18], north [19], northwest [20,21], southwest [22], and south [23] of Iran.
Hyalomma marginatum had the second greatest prevalence in the study area. Additional Hyalomma tick species collected from animals in this study included species of Hy. asiaticum, Hy. dromedarii, Hy. detricum and Hy. schulzei. There have been reports of these species from different parts of Iran [17,24].
The present study contributes new information about the risks of high diverse Hyalomma infestation of domestic animals in south-eastern Iran. This situation may address the animal traffic from neighbouring countries that may lead to more frequent encounters with these tick species. The ticks in this study were feeding on the animals at the time of collection and were therefore potentially transmitting any Anaplasma spp to the animals while feeding. However, the possibility that these tick play a significant role in anaplasmosis transmission to domestic animals where it is endemic requires further investigation. Hyalomma spp ticks in this region carried the Anaplasma and Ehrlichia agents, including A. ovis, A. marginale, and E. ewingii. These Anaplasmataceae pathogens were previously detected using molecular methods in hard ticks in several regions in Iran [11,14,15,25]. The prevalence of Hyalomma spp with Ehrlichia /Anaplasma spp DNA in this study was 68%. This value is higher than the rate of infection in the previous reports from other parts of the country. The prevalence of infection were reported as 4.6% [15], 5.1% [26], 6% [27], 25% [14], 25.8% [10], 26.4% [9], 43.84% [28], and 55.5% [12].
Result of this study and above literature showed that different species of Hyamlomma could be one of the primary carriers and reservoirs for Anaplasma /Ehrlichia spp in the country. In addition to Hyalomma spp ticks, other hard ticks including Rhipicephalous bursa, R. sanguineous, Dermacentor marginaus, Haemaphysalis erinacei, Ixodes ricinus [8,9,15,28] are reported as vector of different tick-borne bacteria of the family Anaplasmataceae. However, in other parts of the world, the most important vector of anaplasmosis belongs to different species of Ixodes genus; for example Ixodes ricinus in Europe, I. persulatus in Eastern Europe and Asia, and I. scapularis in North America [29].
dromedarii collected on goats and camels respectively. Both A. ovis and A. marginale are important livestock pathogens whereas E. ewingii is an important human pathogen. Ehrlichia ewingii mainly infects granulocytes, triggering granulocytic ehrlichiosis in dogs and humans [18,30]. Granulocytic ehrlichiosis in humans has been described in immunosuppressed as well as immunocompetent patients presenting headache, fever, myalgia, vomiting, nausea, acute renal failure, thrombocytopenia, leukopenia and increased liver enzyme activities [31][32][33]. Anaplasma ovis is less pathogenic than other Anaplasma species, has got worldwide distribution, and is responsible mostly for small ruminant anaplasmosis with a low ranking fever [34,35]. However, it may be an important disease agent for sheep and goats [36,37]. Fever, anorexia, fatigue, milk reduction and abortion with a low death rate are the common clinical marks of A. ovis in infected animals [38]. Anaplasma marginale is known as the most important rickettsia disease in cattle. The common clinical signs of the disease are progressive haemolytic anaemia, decrease milk production, abortions, and death. In addition to cattle, other animals including water buffalo, and wild mammals like deer can be infected [39].

Conclusions
In general, farmers and people in border line of Iran-Pakistan who are engaged in livestock need to be made aware of the risks of tick infestation and the tick-borne disease they transmit. Pathogens carried by ticks can infect both animals and humans and monitoring of ticks and the pathogens they carry provides insight into the occurrence and spread of zoonotic diseases. Veterinarians in the region should keep these risks in mind and educate people regarding the risks as well as developing optimal approaches for tick protection protocols that maximize people agreement.  (Fig. 3). The collection of ticks was performed between November, 2017 and March 2018.

Methods
Totally, 1020 samples were randomly collected from goats, sheep, cattle, and camel. Tick collection was arbitrarily conducted based on the availability of domestic animals for 15 minutes per animal, but efforts were made to obtain a widespread representative sample within the different animal species included in the study. All ticks were transferred to vials and labelled according to their origin of geographic and the animals. The collected ticks were referred to the Entomology Laboratory in the School of Public Health at the Tehran University of Medical Sciences and were identified to species level based on morphological characteristics and the method of Estrada-Pena [41].

DNA extraction
After species identification, the ticks were sterilized by immersion in 70% alcohol and washed in distilled water and dried on filter paper in a laminar-flow hood then stored at -80°C until the DNA extraction. DNA extraction was done using the G-spin Genomic DNA Extraction Kit (iNtRON Biotechnology, South Korea) and carried out according to the manufacturer instructions by grinding of individual ticks in an Eppendorf microtube after isolated tick incubation in the liquid nitrogen tank.
The extracted DNA was suspended in sterile distilled water and were then stored at -20°C prior to molecular investigation.

Molecular detection of Anaplasma /Ehrlichia spp
In this study we followed the method of Li et al [42] to differentiate species of Anaplasma genera based upon genetic analyses of 16SrRNA. The Anaplasma/Ehrlichia spp 16SrRNA gene was amplified using the nested PCR protocol and the species-specific primers already designed by Rar et al [43] in (Table 4). The forward and reverse primers for outer reactions were Ehr1and Ehr2 and for inner reactions were Ehr3 and Ehr4. PCR reactions were performed in 25μL reaction mixture containing 12.5μL of the Hot Start Taq 2X Master Mix, 1μL of each of the forward and reverse primers, 2μL of DNA template and 7.5μL of nuclease-free H 2 O to bring the volume to 25μL. PCR reactions were performed in a DNA thermocycler (Eppendorf, Germany) and PCR condition was done to 15 min at 95°C for initial denaturation step, 60 s at 94°C in each cycle for denaturing step, 60 s at 57°C for annealing and 60 s at 72°C for extension step followed by 35 cycles and then a final extension for 10min at 72° C. 2μL of the products of the first round of PCR was used as the template for the second round of PCR, which was carried out under the same conditions and reaction mixture as the first round except that were used as the primers [43].
To assess the presence of specific band for anaplasma spp., PCR amplification was electrophoresed in 1.5% agarose gel and size of each PCR product was estimated using a 100 base pair (bp) ladder run on the same gel as the marker and then visualized under a UV transilluminator. Two negative controls including double distilled water and DNA template of non-infected tick and a positive control (Anaplasma DNA) were included in each PCR assay.

DNA sequencing and Phylogenetic analysis
The positive PCR products were purified and bidirectional DNA sequencing was performed using the same inner PCR primers used for nested PCR amplifications. The acquired sequences in this study were edited and assembled using Chromas and Bioedit softwares to construct consensus sequences and analysed using blast in NCBI (Nucleotide collection) database (https://www.ncbi.nlm.nih.gov/). The consensuses of confident sequences were aligned with other Anaplasma corresponding sequences available in Genbank using multiple-sequence alignments available in CLUSTAL Omega (https://www.ebi.ac.uk/Tools/msa/clustalo). Also available gene sequences of Erlichia ewingii, and Spiroplasma sp as an out group, were obtained from Genbank and combined with the Anaplasm sequences for phylogenetic analysis. All DNA sequences used for alignment were cut to get a consistent region (470 bp). The obtained sequences in the current study were submitted to Genbank (Table 3). Phylogenetic and molecular evolutionary analyses were conducted with MEGA 7 software [44]. For phylogenetic analysis, three representative sequences of A. ovis, one representative sequence of A.marginale, and one representative sequence of E. ewingii [45], obtainedfrom this study were combined with a subset of available representative sequences of all Anaplasma spp and E.
ewingii . Details of the sequences used for this study have been shown in Table 5. The data were aligned and the Maximum likelihood method was employed to construct a phylogenetic tree. The same program was utilized to evaluate the stability of the obtained tree through bootstrap analysis with 1,000 replicates. Availability of data and materials All data generated or analysed during this study are included in this published article.

Competing interests
The authors declare that they have no competing interests.

Funding
This work has been supported financially by Tehran University of Medical Sciences, Iran, grantnumber 29005.
Authors' contributions NC performed the whole study and writing the manuscript, FK accomplished phylogenetic analysis, MK helped in molecular analysis of data, JN was a major contribution in designing and sample collection, and MAO analyzed and interpreted the data, and was a major contributor in editing the manuscript. All authors read and approved the final manuscript.