Circulation of Brucellaceae, Anaplasma and Ehrlichia spp. in borderline of Iran,Azerbaijan, and Armenia

Rahimeh Abdoli, Hasan Bakhshi, Sedigheh Kheirandish, Faezeh Faghihi, Asadollah Hosseini-Chegeni,Mohammad Ali Oshaghi, Zakkyeh Telmadarraiy,5, Mohammad Mehdi Sedaghat✉

1Department of Medical Entomology and Vector Control, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran

2Department of Oral and Maxillofacial Pathology, School of Dentistry, Bushehr University of Medical Sciences, Bushehr, Iran

3Cellular and Molecular Research Center, Iran University of Medical Sciences, Tehran, Iran

4Department of Plant Protection, Faculty of Agriculture, Lorestan University, Khorramabad, Iran

5Rahyan Novin Danesh (RND) Private University, Sari, Mazandaran, Iran

ABSTRACT

KEYWORDS: Tick; Anaplasma; Ehrlichia; Brucellaceae; PCR

1. Introduction

Ticks are one of the most important external blood-sucking parasites of vertebrates that cause the transmission of many pathogens including arboviruses, parasites and bacteria while they suck blood. The most important diseases transmitted by ticks are spotted fever, Rocky Mountains fever, Siberia tick typhus,tularemia, Lyme disease, tick-borne relapsing fever, Crimean-Congo hemorrhagic fever, babesiosis, anaplasmosis and brucellosis[1,2].Ticks can suck blood on mammals, birds, reptiles, and amphibians,while most of them have a different mammalian host at each stage of their lives. These arthropods become infected from an infected vertebrate host and after a while, they are able to transmit pathogens to a new host by blood-sucking activity at another stage of life and the disease cycle remains stable in the nature[3].

Many diseases are transmitted through the bite of ticks: The Brucellaceae family comprises pathogens and soil bacteria with Brucella as an important genus. Brucellosis is caused by different Brucella species which are Gram-negative rod-shaped facultative intracellular bacteria that usually persist for life[4]. Zoonosis species include Brucella (B.) abortus, B. canis, B. melitensis, and B. suis. Unpasteurized milk, undercooked infected meat, close contact with animal secretions are the main routes of transmission to human; on the other hand, ticks usually transmit the disease to sheep, cattle, goats and some other animals[5]. All provinces of Iran are endemic for brucellosis, especially Hamadan, Markazi,Lorestan, Kermanshah, West Azerbaijan, South Khorasan and East Azerbaijan, imposing high economic loss due to livestock infertility and abortions, veterinary expenses and has serious threat to human health[4]. Anaplasmosis is another zoonotic disease caused by obligate intra-cellular bacteria (Family Anaplasmataceae: Order Rickettsiales), which is usually transmitted through a number of hematophagous species of ticks especially Ixodidae family. Species of veterinary importance include: Anaplasma (A.) bovis, A. centrale,A. marginale, A. ovis, A. phagocytophilum (zoonotic) and A. platys[6,7].Anaplasmosis in livestock is characterized by some symptoms including acute fever, anorexia, weight and milk reduction and finally death[8]. Ehrlichia genera is also an obligate intracellular bacterium (Family Anaplasmataceae: Order Rickettsiales) that infect animals and humans[9]. Piroplasmosis caused by Theileria and Babesia species leads to major economic losses to the livestock industry especially in Asia[10]. Piroplasmosis ranges from subclinical to acute with signs including fever, anemia, severe lethargy, and circulatory shock[11,12].

Iran is endemic for many diseases including some arthropodborne diseases. Many studies have been carried out in many provinces, while in some provinces the related investigations seem to be insufficient. One of these provinces is East Azerbaijan. The province shares common borders with the Republic of Azerbaijan and Armenia in the north. In some parts of East Azerbaijan, animal husbandry plays a major role in people’s lives. In addition, the common border with other countries allows the livestock and wildlife trafficking, which plays an undeniable role in transmitting the diseases from one country to another.

This study was performed to estimate the infection of ticks with pathogens Anaplasma, Ehrlichia, Babesia, Theileria and Brucellaceae using molecular methods in Ahar and Kaleybar cities of East Azerbaijan province, north of Iran.

2. Materials and methods

2.1. Study area

East-Azerbaijan is located in northeast of Iran (38.076 6° N 46.280 0° E), bordering with Armenia, Republic of Azerbaijan in north, Ardabil Province in east, West Azerbaijan Province in west,and Zanjan Province in south (Figure 1). East-Azerbaijan covers an area of 47 830 km² and Sahand Mountain lying at south is the highest area (3 707 m), whereas the lower lying areas are around Ahar at north. Climate of East-Azerbaijan is cold mountainous affected by Mediterranean continental.

Figure 1. East Azerbaijan province is located in northwest of Iran. The sampling locations are marked with asterisks.

Figure 2. Evolutionary tree of Anaplasma and Ehrlichia sequences. Data was obtained from this study and similar sequences from other investigations,deposited in GenBank; Wolbachia pipientis included as outgroup. The scale bar indicates an evolutionary distance of 0.01 nucleotides per position in the sequence. Sequences derived from the present study are marked with asterisks. The percentage of trees in which the associated taxa clustered together is shown next to the branches.

Figure 3. Evolutionary tree of Brucellaceae sequence. Data was obtained from this study and similar sequences from other investigations, deposited in GenBank; Escherichia coli included as outgroup. The scale bar indicates an evolutionary distance of 0.02 nucleotides per position in the sequence. The sequence derived from the present study is marked with an asterisk. The percentage of trees in which the associated taxa clustered together is shown next to the branches.

Ahar is located in a mountainous area (38° 28’ N 47° 04’E) limited to Kaleybar from the north, Harris from the south, Varzeqan from the west and Meshkinshahr from the east. The climate is cold mountainous. This city is surrounded by important mountains such as Gheez Qalehsi (1 266 m) and Hashtsar (2 536 m) which are stretched from north to south and west. The lowest point of Ahar is Qarahsu River, which is located in the eastern parts. Kaleybar is also located in a mountainous area (38° 51’N 47° 01’E) which is bordered by the Republic of Azerbaijan, Armenia and Aras River from the north,Jolfa city from the west, Ardabil province (Moghan plain) from the east and Ahar city from the south. Its main mountain range is called Qare Dagh, which has several peaks with different heights such as Gasht Sar(2 940 m), Ghazi Bolagh (2 700 m), Shivar (2 652 m), Sarparq (2 860 m)and Zinghaloo (2 900 m). Kaleybar has a mountainous climate and the whole region is covered with forests and pastures.

2.2. Collection and identification of ticks

A total of 2 022 ticks were collected from different parts of East-Azerbaijan province during spring and summer, 2018 as ticks have their highest activity during these seasons. Multistage random sampling method was used and based on Cochran’s formula (95%confidence level), the sample size required for each molecular assay was determined[13]. Ticks were separated from different body parts including ear, groin, tail, back and neck. Sheep, goat and cow were sampled as animals of interest. There were no specific criteria for animal selection. However, it should be noted that all the animals were apparently healthy and were mostly under 3 years old. Forceps were used with special care not to harm the ticks and animals. Ticks were then placed in appropriately labeled tubes and transferred to the Department of Medical Entomology, School of Public Health,Tehran University of Medical Sciences, Iran along with cold chain for species identification. Stereomicroscope was used to determine genus and spe¬cies according to valid morphological keys[14].

2.3. DNA extraction, molecular detection of pathogens and sequencing

DNA was extracted using Exgene DNA extraction kit (GeneAll,Korea) according to the manufacturer’s instructions. A 20 μL reaction mixture containing 10 mm Tris-HCl (Ph 9.0), 30 mm KCl,1.5 mm MgCl, 250 mm dNTPs, 0.5 mm each sense and antisense primers, 1 U Taq DNA polymerase and 2 μL of DNA were used for PCR. PCR thermal program was as follows: Initial denaturation at 94 ℃ for 5 min followed by 35 cycles of amplification (1 min denaturation at 94 ℃, 1 min annealing at 57 ℃ and 1 min elongation at 72 ℃[15]. A 524 bp fragment of the 16S rRNA gene of Anaplasma and Ehrlichia was amplified by nested-PCR[16]. 18S rRNA was targeted to amplify Babesia sp./Theileria sp. genome[16]. Finally, 16S rRNA of Brucellaceae genome was targeted for the production of 712 bp amplicons[17]. Sets of primers and their characteristics for each PCR reaction is listed in Table 1. Genomic DNA of Anaplasma,Ehrlichia, Babesia, Theileria and Brucella species were obtained from the Department of Parasitology, Faculty of Veterinary Medicine,University of Tehran, Iran and used as positive controls. Double distilled water was served as negative controls. The PCR analysis was performed on 1.5% agarose and UV condition. Among the positive samples of each species, the samples with a sharp band of the expected size were chosen for sequencing to confirm the accuracy of the PCR reaction. Sequencing of selected PCR products was performed by Takapouzist Company, Iran. Sequences were checked to correct any sources of error using Chromassoftware.The nucleotide sequences were then blasted by BLASTN (http://www.ncbi.nlm.nih.gov/BLAST) program.

Table 1. Details of the primers, target gene and product size of PCR assays.

Table 2. Prevalence of ticks in respect to sex, life stage and sampling season.

2.4. Statistical analysis

SPSS V19 (IBM, USA) was used for statistical analysis.Descriptive statistics were used to summarize the variables. Also 95% confidence interval for prevalence of tick infestation was calculated using binomial distribution (significant level less than 0.05).

3. Results

3.1. Identification and characteristics of ticks

Out of a total of 1 400 livestock (sheep, goat, buffalo and cattle),2 022 hard ticks were collected. Four genera and 9 species were morphologically identified including: Hyalomma (Hy.) anatolicum:39.21% (95% CI 37.08%-41.39%), Hy. marginatum: 9.14% (95% CI 7.93%-10.49%), Hy. asiaticum: 0.69% (95% CI 0.38%-1.16%), Hy.nymph: 38.67% (95% CI 36.55%-40.84%), Hyalomma sp.: 1.03%(95% CI 0.76%-1.76%), Hy. larva: 0.04% (95% CI 0.00%-0.28%),Rhipicephalus (Rh.) bursa: 5.19% (95% CI 4.27%-6.25%), Rh.sanguineus: 3.61% (95% CI 2.84%-4.52%), Haemaphysalis (Hae.)sulcata: 0.59% (95% CI 0.31%-1.03%) Hae. concinna: 0.04% (95%CI 0.00%-0.28%), Hae. punctate: 0.14% (95% CI 0.03%-0.43%) and Dermacentor marginatus: 1.58% (95% CI 1.09%-2.23%). The rate of tick collection in spring and summer was 13.10% (95% CI 11.66%-14.65%) and 86.89% (95% CI 85.35%-88.34%), respectively. A total of 498 males [24.62% (95% CI 22.76%-26.57%)], 741 females[36.64% (95% CI 34.54%-38.79%)], 782 nymphs [38.67% (95%CI 36.55%-40.84%)] and 1 larva [0.04% (95% CI 0.00%-0.28%)]were identified (Table 2). Totally, 1 666, 316, 22 and 18 ticks were collected from cattle, sheep, goats and buffalos, respectively.

3.2. Molecular detection

Table 3 summarizes the data related to tick species which were examined for detection of Anaplasma, Ehrlichia, Babesia, Theileria and Brucellaceae using nested PCR assay for detection of a 524 bp fragment of Anaplasma, Ehrlichia spp. 16S rRNA indicated that Hy.asiaticum, Hae. sulcata, Hy. anatolicum, Rh. bursa, D. marginatus,Hy. marginatum and Rh. sanguineus were positive. Amplificationof a 712 bp fragment of 16S rRNA from Brucellaceae genome also revealed that Hy. asiaticum, Hae. sulcata, Rh. bursa, D. marginatus and Hy. marginatum were positive. On the other hand, no genomic material from Babesia spp./Theileria spp. was detected in tested samples (Table 3).

Table 3. Detail of No. of species examined using PCR for detection of parasites.

3.3. Sequencing and phylogenetic analysis

A total of 6 sequences with a sharp band of the expected size were submitted and the accession numbers MH538092 (Anaplasma sp.),MH538094 (Ehrlichia sp.), MH538095 (Anaplasma sp.), MH538096(Anaplasma sp.), MH538097 (Anaplasma sp.), MH538098(Anaplasma sp.) and MH538966 (Brucellaceae) were assigned in GenBank. Submitted sequences were aligned using Clustal-W algorithm. The evolutionary tree of Anaplasma and Ehrlichia sequences obtained from the present study (MH538092, MH538094,MH538095, MH538096, MH538097, and MH538098) and similar sequences from other investigations, deposited in GenBank was inferred by using the maximum likelihood method with bootstrap of 1 000 replications[18,19](Figure 2). Furthermore, the evolutionary tree of Brucellaceae sequence obtained from this study (MH538966) and similar sequences from other investigations, deposited in GenBank was inferred using the maximum likelihood method with bootstrap of 1 000 replications[18,19](Figure 3).

Initial trees for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood approach, and then selecting the topology with superior log likelihood value. Trees were drawn to scale, with branch lengths measured in the number of substitutions per site[19]. The results of phylogenetic analysis revealed that three identified Anaplasma sp. had the most similarity with A. centrale (Accession Number: AF283007.1). Two other detected Anaplasma sp. had the most similarity with A. platys, A. camelii and A. phagocytophilum(Accession Numbers MG050139.1, KX765882.1, and KF002508.1 respectively). Further to detected Ehrlichia sp., it has the most similarity with Ehrlichia ruminantium (Accession number:NR074513.2) (Figure 2). Furthermore, the detected Brucellaceae bacterium had the most similarity with Mycoplana peli (Accession number: LC177126.1).

4. Discussion

Ticks are considered as important vectors of pathogens which play a critical role in the survival and transmission of the pathogens that cause diseases in humans and animals[20]. In the present study, Hy.anatolicum was determined as the dominnat specie and Hyalomma as the dominant genera. Conducted studies in north in Hamadan, West-Azerbaijan and Ardabil provinces showed that Hyalomma genera was reported as the predominant genus of collected ticks[21-24]. It should be also noted that in many northern provinces, Rhipicephalus spp. were determined as the dominant species[25-27] and in some studies, both of the genera were considered to be dominant[28]. In the current investigation, four genera were detected at the Azerbaijan-Armenia borderline in Iran, including Hyalomma, Rhipicephalus,Dermacentor and Haemaphysalis. All of these genera have been reported to be incriminated for transmission of Crimean Congo hemorrhagic fever virus in different countries including Iran[29]. The detected tick genera in the present study have also been considered important from the medical and veterinary perspective[30]. The most infected hosts in terms of tick infestation were cattle and most of ticks were collected in the summer which indi¬cate higher activity of ticks in hot temperature. Furthermore, 37 out of the 168 (22%) tested ticks were positive for the Anaplasma, Ehrlichia spp. including Hy.asiaticum, Hae. sulcata, Hy. anatolicum, Rh. bursa, D. marginatus,Hy. marginatum and Rh. sanguineus. Tajedin et al. reported tick infection with Anaplasma or Ehrlichia spp. across East-Azerbaijan(71%) which is higher than our result[31]. This difference can be justified due to larger sample size and host variability in the present study. Jafar-Bekloo et al., indicated the rate of tick infection with Anaplasma and Ehrlichia spp. using molecular assays in north of Iran: 25% of ticks were infected including: R. sanguineus, R. bursa,Hy. marginatum and Hy. scupense, which is in concordance with our study except that Hy. scupense was not detected in the present study[6]. In a molecular study on Anaplasma and Ehrlichia spp.identification in Hyalomma ticks in border line of Iran-Pakistan, it was indicated that Hy. anatolicum was the most prevalent specie (in line with the present study) and Anaplasma or Ehrlichia spp. were detected in 68.3% of the specimens[32]. The higher prevalence might be due to the hot and dry climate of the Iran-Pakistan border, the greater rate of traditional animal husbandry in the region and the lack of awareness of animal health principles. A 16S rRNA gene fragment of Anaplasma species was identified in 26.4%, 49.5%, 58.3% and 59% of tested ticks at Afghanistan borderline, Mazandaran, Kerman and Savadkooh regions of Iran, respectively[9,15,33,34]. Further to the previous studies, it can be concluded that in most parts of Iran,Rhipicephalus spp. are the most infected ticks to Anaplasma spp. and Ehrlichia spp.

The role of Argasidae and Ixodidae ticks in preservation and transmission of Brucellaceae in nature and from one animal to another have already been speculated. Previously, the presence of B.abortus in lice of several ruminants was reported[35-37]. To the best of our knowledge, few molecular studies have been performed on the infection of ticks with Brucellaceae family in Iran and other parts of the world. A Brucella-like bacterium was detected in a Boophilus tick using PCR method. Ticks were collected from Talesh County,Guilan Province, north of Iran[17]. Molecular detection of tickborne pathogens harbored by ticks collected from livestock in the Xinjiang Uygur Autonomous Region, China revealed that 26.2% of tested ticks were infected with Brucella sp.[38]. B. melitensis and B.abortus were also detected in eggs, larvae and engorged females of D. marginatus collected from sheep and goats from different parts of China[39]. In the present study, the Brucellaceae genome fragment was detected in Hy. asiaticum, Hae. sulcata, Rh. bursa, D. marginatus and Hy. marginatum. The members of the genera Hyalomma,Dermacentor, Rhipicephalus and Haemaphysalis seem to act as vectors of Brucella spp. in nature. However, further studies seem necessary to confirm the result.

No Babesia sp./Theileria sp. genome was found in the collected ticks which may suggest that ticks don’t have a significant role in the circulation of these pathogens in the study area. Other studies from other parts of Iran, however, have shown various results in terms of tick infection with Babesia sp./Theileria sp. Molecular surveillance of Theileria (T.) ovis, T. lestoquardi and T. annulata infection in sheep and ixodid ticks in Razavi-Khorasan province, north east of Iran revealed that 9% of R. turanicus were positive for Theileria species[40]. Hasheminasab and colleagues reported that Rhipicephalus spp. and Hy. anatolicum had the highest infection rate with Babesia and Theileria in Dehgolan, Iran[41]. Molecular detection of Theileria spp. in tick vectors in Fasa and Kazeroun areas of Iran also revealed that one pool of H. turanicus was infected with T. ovis[42]. Habibi et al. showed 18 isolated tick DNAs (66.7%) from dogs in Shahriar County, Iran were infected with T. annulata[43]. Tick infection with Theileria and Babesia in Iran renges between 6.1% to 55% and 6.25% to 76%, respectively[6,44-48]. It seems that differences between the results of the present study and similar studies in other regions of Iran is due to differences in the sampling season, sample size,climate conditions, mollecular methods, livestock management and husbandry. It is recommended that anaplasmosis, ehrlichiosis as well as brucellosis should be considered important health issues in northwest of Iran and consistent monitoring on tick vectors and livestock should be performed regularly. It is also suggested to conduct the study in autumn and winter seasons on vertebrate hosts (especially domestic animals) and the people who are at risk (veterinarians and ranchers) to clarify the status of diseases more accurately.Boundaries must be carefully monitored and the transportation of animals, even wild animals, must be closely observed.

In the present study, we detected the infection of tick with Anaplasma sp./Ehrlichia sp. and Brucellaceae. It can also be concluded that in the north and north-west of the country, Hyalomma and Rhipicephalus species are the most prevalent and important species among hard ticks.

Conflict of interest statement

The authors declare no conflict of interest.

Authors’ contributions

Both R.A. and H.B. contributed to performing the experiments and contributed to the final version of the manuscript. S.K., F.F., A.H.C and M.A.O. analyzed the data and edited the manuscript. Both Z.T. and M.M.S. supervised the project and contributed to the final version of the manuscript. All authors reviewed the manuscript.