Anaplasma spp. identification in hard ticks of Iran: First report of Anaplasma bovis in Haemaphysalis inermis

The aim of the present study was to determine the presence of Anaplasma spp. in hard ticks in the north of Iran. Tick samples were collected from sheep and goats grazing in Savadkooh, Mazandaran province and identified under a stereomicroscope according to identification keys. Salivary glands of the ticks were dissected and a polymerase chain reaction (PCR) assay, followed by partial sequencing of the 16S ribosomal RNA gene, was used for the detection and identification of Anaplasma spp. in the DNA extract of the salivary glands. A total of 618 ticks were collected from 122 sheep and goats from Savadkooh. The identified tick specimens belonged to 5 genera and 11 species including Rhipicephalus bursa, Rh. sanguineus, Rh. turanicus, Rh. (Boophilus) annulatus, Haemaphysalis punctata, Ha. concinna, Ha. parva, Ha. inermis, Hyalomma marginatum, Dermacentor marginatus and Ixodes ricinus. Eight of these, including Rh. sanguineus, Rh. bursa, Ha. punctata, Ha. inermis, Ha. concinna, D. marginatus, Rh. turanicus and I. ricinus, were positive for the presence of Anaplasma. All of the sequenced samples showed 99-100 % identity to Anaplasma bovis. The present paper is the first to detect A. bovis in Rh. sanguineus, Rh. bursa, Ha. punctata and D. marginatus in Iran; the highest infection rate of A. bovis in the collected ticks was found in Rh. bursa. This research is also the first report of A. bovis in Ha. inermis in the world.


INTRODUCTION
Ticks are blood sucking ectoparasites that play a significant role in the transmission of many pathogens to both animals and humans throughout the world. Anaplasma is a bacterial genus that includes several tick-borne pathogens causing anaplasmosis in animals and humans. Anaplasma spp. are small gram-negative obligate intracellular organisms. The genus Anaplasma includes A. marginale, A. centrale, A. phagocytophilum, A. bovis, A. ovis and A. platys (Rymaszewska and Grenda, 2008;Ybanez et al., 2014). A. marginale is the main intraery-throcytic agent of bovine anaplasmosis. A. centrale is less pathogenic than A. marginale. A. phagocytophilum tends to invade and propagate in polymorphonuclear leucocytes causing human granulocytic anaplasmosis (HGA), tick-borne fever (TBF) in ruminants, and canine and equine granulocytic anaplasmosis (Rymaszewska and Grenda, 2008). A. bovis mainly occurs in monocytes of cattle but also has been detected in small ruminants, dogs, cats, rabbits and wild mammals which are probably reservoirs of the bacterium. Infection in cattle is usually asymptomatic but can result in clinical signs including fever, anemia, weight loss and enlargement of prescapular lymph nodes. (Uilenberg, 1997;Goethert and Telford, 2003;Santos and Carvalho, 2006;Sakamoto et al., 2010;Liu et al., 2012;Sasaki et al., 2012;Said et al., 2015). A. ovis is an intraerythrocytic pathogen of sheep, goats and wild ruminants (de la Fuente et al., 2004) and is less pathogenic in sheep than in goats. There is a paucity of information about the tick vectors of these rickettsial agents in Iran (Donatien and Lestoquard, 1936;Bashiribod, 2004;Hosseini-Vasoukolaei et al., 2014;Saghafipour et al., 2014). The aim of the present study is thus to determine the presence of Anaplasma spp. in the salivary glands of hard ticks collected from grazing sheep and goats of Savadkooh in Mazandaran province.

Study area
Savadkooh is located in the south of the Caspian Sea with an altitude range of 250 m to 3651 m above the sea level. This region has a short summer with a mild, humid Mediterranean climate and long, freezing cold winters. Changes in altitude and slope result in high variation in weather and vegetation conditions. Vegetation up to 2000 m above sea level is green and forested, whereas above this altitude, there is only low vegetation with arid and cold weather. There are different types of trees, shrubs and grasses in the different areas with a variety of animals using the landscape: cattle, sheep, goats and deer feed and graze in this region, although sheep outnumber the other livestock species.

Tick sampling and identification
The tick samples were collected by examining the sites of predilection for ticks on the bodies of 86 sheep and 36 goats during the first 6 months of 2012. These animals belonged to 10 separate herds located across in the region. Individual ticks were counted on the animals and preserved in separated vials containing 70 % ethanol. Adult ticks were identified under a stereomicroscope according to identification keys (Walker et al., 2003;Estrada-Peña et al., 2004). Salivary glands of the ticks were dissected according to Edward et al. (2009). For each tick, sterilized scalpel blades were used to avoid possible contamination.

DNA extraction and PCR
Total DNA was extracted from the individual salivary glands of each tick using a DNA extraction kit (MBST, Tehran, Iran) and following the manufacturer's instructions.
The presence of Rickettsiales of the genus Anaplasma was assessed based on the presence of the 16S rRNA gene by PCR as previously reported (Noaman et al., 2009). Two primer pairs that cover the hypervariable region of this locus were used. A first amplification was performed using primers F2 (5'-agagtttgatcctggctcag-3') and R2 (5'-agcactcatcgtttacagcg-3'). To control the specificity of the PCR products, a nested PCR was then performed to amplify an internal 543bp fragment of the same gene using a second pair of primers (F3=5'-gcaagcttaacacatgcaagtc-3' / R3=5'gttaagccctggtatttcac-3'). These primers were designed by Noaman et al. (2009).
Approximately 20 ng of DNA was used for the PCR analysis performed in 100 µL total volume including 10x PCR buffer, 2.5 U Taq Polymerase (Sinaclon, Iran), 2 µL of each primer (20 µM, Sinaclon, Iran), 2 µL of each dATP, dTTP, dCTP and dGTP, (100 µM, Fermentas), 1.5 mM MgCl 2 (50mM, Sinaclon, Iran). Reactions were carried out in automated thermal cyclers (Bio-Rad) with the following program: 5 min incubation at 95°C to denature doublestranded DNA, followed by 35 cycles of 45 s at 95°C (denaturation), 45 s at 59°C or 55°C (annealing) and 45 s at 72°C (extension) and an additional extension step at 72°C for 5 min. As a positive control, DNA extracted from A.marginale was used. As a negative control, we used distilled water. The annealing temperature for the PCR reaction was 50°C. Amplified PCR products were analyzed by electrophoresis on 1.5 % agarose gels, stained with Cybersafe and visualized under UV light. The PCR products were purified using a PCR purification kit (MBST, Tehran, Iran) and were directly sequenced by Kowsar Company (Iran, Tehran). For the analysis, the obtained nucleotide sequences were input to the Basic Local Alignment Search Tool (BLASTn) on the National Center for Bio-technology Information (NCBI) database website.
A phylogenetic tree was constructed with MEGA 6 software, applying the UPGMA method with bootstrap analysis (1,000 replicates).

RESULTS
In the present research a total of 618 ticks (294 female and 324 male) were collected from 122 sheep and goats. The identified tick specimens belonged to 5 genera and 11 species including Rhipicephalus bursa (27 %), Ha. punctata (27.5 %), Rh. sanguineus (3 %), Ha. concinna (7 %), Rh. turanicus (24 %), Ha. parva (3 %), Hy. marginatum (2 %), D. marginatus (2 %), I. ricinus (2 %), Ha. inermis (2 %) and Rh(B). annulatus (0.3 %). Amplification of the 18S rRNA gene of the ticks showed that DNA extracted from the salivary glands was of good quality. The amplicons obtained from the nested-PCR for Anaplasma were approximately 781bp and 543bp, respectively (Fig.1). The  The similarity among the sequenced strains of Anaplasma in this study was 100 % except for A. bovis from D. marginatus showing 99.8 % identity with the others. In this investigation, no infection was detected in the following species: Ha. parva, Hy. marginatum and Rh (B). annulatus. Rh. turanicus and I. ricinus were positive in PCR and nested-PCR for the presence of Anaplasma, however, the Anaplasma sequences obtained from these tick species could not be identified due to incomplete sequencing. Our results showed that the most infected ticks were Rh. bursa and Rh. turanicus with 72 and 65 % infection rate respectively.
A phylogenetic tree based on the similarity between our sequences with registered sequences in GenBank showed 2 subclades for Anaplasma spp.: one subclade including A. platys, A. phagocytophilum and A. bovis and the other with A. marginale, A. ovis and A. centrale. Wolbachia and Ehrlichia were in separate clades (Fig.2).

DISCUSSION
Ticks transmitting pathogens, such as the Crimeancongo haemorrhagic fever virus, Anaplasma spp., Ehrlichia spp., and Babesia spp. are serious threats to human and animal health. As global warming can result in climate change and modifications in the distribution of tick species and tick-borne disease agents (Aydin and Bakirci, 2007), regular monitoring of tick species and associated infectious agents is essential for understanding disease risk and for implementing control and prevention strategies.
A. bovis was first described in Iran (Donatien and Lestoquard, 1936) and this was followed by reports in many other countries such as Africa, Brazil, North America, China, Japan and Korea (Goethert and Telford, 2003;Kawahara et al., 2006;Ooshiro et al., 2008;Liu et al., 2012;Doan et al., 2013). Anaplasmosis has been reported to be present in animals in Iran by many investigators (Spitalska et al., 2005;Razmi et al., 2006;Ahmadi-Hamedani et al., 2009;Noaman and Shayan, 2009;Noaman et al., 2009;Noaman and Shayan, 2010;Jalali et al., 2013); however, there are few studies about the tick vectors of this rickettsial agent in the country. In the present work, the 16S rRNA gene was employed as a sensitive molecular tool for the detection of Anaplasma DNA (Kang et al., 2011). Unlike other studies which typically used DNA from whole ticks or tick pools, we used extracts from individual tick salivary glands in order to quantify prevalence more exactly. In this study A. bovis was detected in all of the collected tick species except Ha. parva, Hy. marginatum and Rh (B). annulatus. Although one paper reported I. ricinus as a vector of A. phagocytophilum in Iran (Bashiribod, 2004), A. phagocytophilum was not detected in this study. Hosseini-Vasoukolaei et al. (2014) showed A. ovis in Rh. sanguineus and I. ricinus and A. bovis in Rh(B).annulatus from Ghaemshahr, Iran (Hosseini-Vasoukolaei et al., 2014). We did not detect Anaplasma spp. in collected Rh. (B) annulatus, but this may be due to the low number of samples of this particular tick species. Saghafipour et al. (2014) isolated A. ovis from Rh. sanguineus in Qom, Iran, but they could not detect any Anaplasma spp. in Hy. dromedarii, Hy. schulzei and Hy. marginatum (Hosseini-Vasoukolaei et al., 2014;Saghafipour et al., 2014). Our results supported the data reported previously by Hosseini-Vasoukolaei et al. (2014) and Saghafipour et al. (2014) dealing with the absence of any Anaplasma spp. in Hy. marginatum. Pazhoom F. et al. According to Hosseini-Vasoukolaei et al. (2014), 43 % of sheep were positive for the presence of Anaplasma spp (A. ovis and A. bovis) in Ghaemshahr that is close to Savadkooh. However, A. bovis is mainly associated with cattle. In our study was also detected this bacterial species in ticks infesting small ruminants, suggesting that sheep and goats could be reservoirs for Anaplasma in cattle.
In conclusion, the present research is the first report of A. bovis in a wide range of different tick species feeding on sheep and goats in Iran. Moreover, the present study has detected the presence of A. bovis in Ha. inermis for the first time in the world. Although the examined ticks were collected from small ruminants rather than cattle, these hosts may function as reservoirs for A. bovis, not only for cattle but also for the wild animals living in the studied areas.