Molecular Investigation and Phylogenetic Analysis of Anaplasma spp. and Ehrlichia spp. (Based on 16S rRNA gene) Isolated from Hyalomma Ticks in the Border of Iran and Pakistan

Background: Anaplasmosis and Ehrlichiosis are tick-borne diseases affecting human beings and livestock in tropical and subtropical regions. Animal husbandry is the main activity of people in the border of Iran and Pakistan, where thousands of cattle cross the borders into the countries weekly. Methods: PCR-direct sequencing of the 16S rRNA gene was used to determine the prevalence and geographical distribution of the disease agents in Hyalomma hard ticks. 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 DNA prevalence in the infected ticks were 81.82, 9.09, and 9.09 % respectively. The DNA sequence and phylogenetic analysis of the 16S rRNA gene conrmed the detection of these three Anaplasmosis agents, showing 99-100% identity with the strains previously reported in Genbank from different part of the world. Conclusions: Since A.ovis and A.marginale are considered as important livestock pathogens, and E.ewingii is an important human pathogen, both farmers and people involved in livestock along the border of the two countries need to be made aware of the risks posed by tick infestation and the tick-borne disease.

Iran has various climates and each one may harbour several different tick species, which is responsible for the difference in the epidemiology of Anaplasmosis/Ehrlichiosis in different regions. The Sistan and Baluchistan province in the southeast corner of Iran has a long border with Pakistan and Afghanistan where infectious diseases do not respect international boundaries. Animal husbandry is one of the main activities of people in the province. Every week thousands of livestock including sheep, goats, cows, and buffaloes cross the borders into the countries. It is suggested about one million animals are traded or tra cked yearly to Iran [5].
Nested polymerase chain reaction (PCR) has been displayed to be a highly sensitive and speci c test for the diagnosis of Anaplasmosis/Ehrlichiosis [6]. Among different markers, the 16S rRNA gene is one of the common markers used for bacterial genotyping [3].
Despite the identi cation of Anaplasma/Ehrlichia species in livestock based on the molecular assays, less study has been performed in their vectors. There have been only a few studies on the detection of tick Anaplasmosis/Ehrlichiosis infections in Iran, which reported Anaplasma/Ehrlichia infection in ticks in the north and other parts of Iran [7][8][9].
Ixodid ticks play an important role in maintaining Anaplasma/Ehrlichia species in nature. It has been reported that Ixodes Latreille , 1795, Rhipicephalus Koch, 1844, Dermacentor Koch, 1844, and Amblyomma Koch, 1844 genera are the main vectors of Anaplasma/Ehrlichia bacteria in different regions of the world [10]. Although it was reported that Hyalomma ticks are proven vectors of A. marginale [11], Hyalomma spp., are known as the main vector of Crimean-Congo hemorrhagic fever [12] and 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. In this study, we identi ed six different Hyalomma species of tick infesting on various domestic animals and further investigated Anaplasma/Ehrlichia infection status in Hyalomma ticks from Sistan and Baluchistan of Iran.  Fig. 1). The collection of ticks was performed in three randomly selected major husbandry farms in each district between November 2017 and late March 2018 when adult ticks are most active in the region.

Sample collection and tick identi cation
Totally, 1020 samples were randomly collected from goats, sheep, cattle, and camels. 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 geographical origin and the animals from which they were obtained. The collected ticks were subsequently transferred to the Entomology Laboratory in the School of Public Health at the Tehran University of Medical Sciences and were identi ed to species level based on morphological characteristics and the method of Estrada-Pena [13].

Dna Extraction
After species identi cation, the ticks were sterilized by immersion in 70% alcohol and washed in distilled water and dried on lter paper in a laminar-ow hood then stored at − 80°C until the DNA extraction. The 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 individual ticks in an Eppendorf microtube after isolated tick incubation in the liquid nitrogen tank. Afterward, the extracted DNA was suspended in sterile distilled water and was 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 [14] to differentiate species of Anaplasma genera based upon genetic analyses of 16S rRNA. The Anaplasma/Ehrlichia spp., 16S rRNA gene was ampli ed using the nested PCR protocol and the species-speci c primers already designed by Rar et al [15]. The forward and reverse primers for outer reactions were Ehr1 (5' -GAA CGAA CGC TGG CGG CAA GC-3') and Ehr2 (5'-AGT A(T/C)C G(A/G)A CCA GAT AGC CGC-5') and for inner reactions were Ehr3 (5'-TGC ATA GGA ATC TAC CTA GTA G-3') and Ehr4 (5'-CTA GGA ATT CCG CTA TCC TCT-3'). 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 nal extension for 10min at 72° C. 2µL of the products of the rst 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 rst round except that were used as the primers [15].
To assess the presence of speci c bands for Anaplasma spp., PCR ampli cation was electrophoresed in 1.5% agarose gel and the 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 positive control (Anaplasma DNA) were included in each PCR assay.

Dna Sequencing And Phylogenetic Analysis
The positive PCR products were puri ed and bidirectional DNA sequencing was performed using the same inner PCR primers used for nested PCR ampli cation. The acquired sequences in this study were edited and assembled using Chromas and Bioedit software to construct consensus sequences and analysed using blast in NCBI (Nucleotide collection) database (https://www.ncbi.nlm.nih.gov/). The consensuses of con dent 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 Ehrlichia ewingii, and Spiroplasma sp., as outgroup, were obtained from Gen bank and combined with the Anaplasm sequences for phylogenetic analysis. All DNA sequences used for alignment were cut to get a consistent region (470 bp). Phylogenetic and molecular evolutionary analyses were conducted with MEGA 7 software [16]. For phylogenetic analysis, three representative sequences of A. ovis, one representative sequence of A.marginale, and one representative sequence of E. ewingii, obtained from 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 1. 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 1000 replicates.   Anaplasma / Ehrlichia infection in ticks Using broad-spectrum EHR primers, 256 out of 1020 collected ticks (25%) were tested for the presence of Anaplasma's or related species 16S rRNA gene. The result of PCR assays revealed the 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 3. The rate of Anaplasma spp., and E.ewingii infections was similar (67.8%-69.2%) in Hy. anatolicum, Hy. dromedarii, Hy. asiaticum, and Hy. marginatum specimens. This rate, however, was higher in Hy. detricum (2 out of 2, 100%) and lower in Hy. schulzei (2 out of 5, 40%) than the other four species. A sub set 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 4.

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 to 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. Similarly, the isolated strains of E. ewingii obtained in this study 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 Gen bank were more than 99-100%.
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 including I) 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 rst comprehensive study of Hyalomma ticks attached to domestic animals and their associated Anaplasma/Ehrlichia species conducted on the border of Iran and Pakistan, southeast corner of Iran. The results show that there are six infesting Hyalomma spp., ticks and that there are at least three Anaplasmosis agents that can be transmitted through a tick bite.
These infestations may cause considerable blood losses from livestock and can also transmit severe tick-borne diseases in the region. These diseases impose massive losses on the livestock sector including reducing the production of meat, milk, wool, and skin in the south corner of Iran. Results of this study is generally in accordance with observations on hard ticks and pathogens from animals in Pakistan neighbouring country and other parts of Iran, while there are some distinctive results in this study [7,17,22].
In this study, Hy. anatolicum-infested animals were found to be the most common and have the widest geographical range. This three-host tick species was previously reported as the most prevalent hard tick from different parts of Pakistan including Baluchistan of Pakistan the closest area to Baluchistan of Iran, and most parts of Iran [7,8,17,18,19,20].
The present study provides new information about the risks of high diverse Hyalomma infestation of domestic animals in south-eastern Iran. This situation may address the animal tra c from neighbouring countries, which 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 ticks play a signi cant 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 [17,19,20,21]. The prevalence of Hyalomma spp., with Ehrlichia /Anaplasma spp., DNA in this study was 68%, which is higher than the rate of infection in the previous reports from other parts of the country. The prevalence of infection was reported as 4.6% [17], 25% [22], 43.84% [23], and 55.5% [9]. The different results regarding the tick infection rate in the study region could be explained by the different environmental factors, collection time, the sampling method, the tick community structure.  [4,34]. Granulocytic Ehrlichiosis in humans has been described in immunosuppressed as well as immunocompetent patients, causing headache, fever, myalgia, vomiting, nausea, acute renal failure, thrombocytopenia, leukopenia and increased liver enzyme activities [25][26][27]. 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 [28,29]. However, it may be an important disease agent for sheep and goat [30,31].
Fever, anorexia, fatigue, milk reduction and abortion with a low death rate are the common clinical marks of A. ovis in infected animals [32] . 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 [33].
The present study revealed circulation of three species of Anaplasmatacea along the border of Iran and Pakistan. This nding is in agreement with the results of researchers on the border of Iran and Afghanistan as well as Pakistan who reported different tick-borne pathogens including Ehrlichia and Anaplasma in ticks [8,18]. In Pakistan, researchers reported A. marginale, A. centrale, A. ovis, A. platys-like organism, E. minasensis, and two uncharacterized species: Ehrlichia sp., Multan and Anaplasma spp., (BL099-6).
In the current study we did not nd A. phagocytophilum or other human granulocytic ehrlichiosis (HGE) agent in the tick specimens. Using more sensitive methods such as real-time PCR (RT-PCR) against ticks or the animal blood of tick hosts might reveal better picture of anaplasmosis agent in the region. Anaplasma phagocytophilum can infect various animals including goats and cows, and can be transmitted to humans by a bite from an infected tick [4].

Conclusions
In general, farmers and people who are involved in livestock farming along the border of Iran and Pakistan need to be made aware of the risks posed by tick infestation and the tick-borne disease.
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's agreement.