Seroprevalence of human Anaplasma phagocytophilum in Bursa Province , Turkey

The aim of this study was to determine the seroprevalence of Anaplasma phagocytophilum infections in rural areas of Bursa Province, where vector ticks and blood-sucking flies are commonly found. In total, 150 blood samples were collected from people living in four different locations where vector ticks are common, and 45 blood samples were collected from people living in non-tick-infested areas. Giemsastained blood smears were prepared from the blood samples and examined by light microscopy. The blood samples were also serologically examined using the indirect fluorescent antibody test (IFAT). One sample (0.7%) from a patient with a tick bite history was positive, as determined by the IFAT; however, the same sample was negative when its Giemsa-stained smear was examined. The co-seroprevalence of A. phagocytophilum and Borrelia burgdorferi was not observed.


INTRODUCTION
Human granulocytic anaplasmosis (HGA) is a zoonotic infection transmitted by ticks to vertebrate hosts.Anaplasma phagocytophilum is an obligate intracellular bacterium that is related to rickettsial organisms and that replicates within the hostile environment of neutrophils (Carlyon and Fikrig, 2003).Acute anaplasmosis do not have specific symptoms; its symptoms are flu-like, such as myalgia, headache and tiredness, and can occur similar to viral diseases.Acute anaplasmosis can cause acute renal failure, haemorrhage, and rhabdomyolysis and can cause death in some cases if treatment is delayed, depending on the host's immunological condition (Lotrič-Furlan et al., 2006).The HGA agent infects the cytoplasm of circulating leukocytes, causing intracellular clumps known as morulae, which are most likely to be found during the first week of illness.The detection test has high specificity but low sensitivity.The presence of morulae in stained blood, bone marrow or cerebro-spinal fluid (CSF) leukocytes is an evidence of the presence of Anaplasma infection, but not for specific species, and supports the HGA diagnosis (Baken and Dumler, 2008).The first case of HGA was identified in the United States of America in 1994 from a patient with febrile illness (Chen at al., 1994).Since then the annual HGA incidence has been gradually increasing in the United States and in average, more than 600 cases were reported annually from 2004-2006 by the Centers for Disease Control and Prevention (CDC, Centers for Disease Control and Prevention, 2008).Local cases have also been reported in Europe (Koebel et al., 2012) (Dumler et al., 2001).Nearly half of the patients with severe HGA require hospitalization with 17% requiring admittance to intensive care (Baken and Dumler, 2008).Ixodes ricinus is a common tick, particularly in the northern regions of Turkey, which are rainy and covered with forest areas.In Bursa Province, I. ricinus (23.49%) is also the most common tick found on ruminants (Aydın, 2000).Therefore, detecting the seroprevalence of Anaplasma infections was the aim of this study.

Study area
Bursa Province is along the coastline of the South Marmara region of Turkey (Coordinates: 40°11′N 29°03′E/40.183°N29.067°E).This province has a moderate climate, with an average rainfall of 16.1-109.6kg/m 2 , the majority of which falls during the winter season.The average temperatures are 23.6°C in summer and 6.2°C in winter.Additionally, the temperature can drop below zero in winter.Mountains cover approximately 35% of the land and are generally in the form of mountain chains running from east to west.The annual temperature, rainfall distribution, average humidity levels and forest-covered areas are suitable for vector ticks (Çalişkan, 2012).

Sampling and parasitology examination
Four villages where I. ricinus ticks have been reported previously were selected for this study.These are Osmangazi-Gökçeören, Orhaneli-Koçuköy, Erenler and Keles-Alpagut villages.In total, 150 blood samples were taken from people living in these 4 villages (71 male, 79 female; ages: 15-87).Additionally, 45 blood samples were collected from people living in areas without the risk of tick infestation (39 male, 6 female; ages: 20-59).The participants were surveyed to identify risk factors before blood samples were collected.Age, gender and a history of tick bites were recorded for each individual.Serum samples were kept at -20°C in a freezer.The blood samples were collected in EDTA containing tubes and were used to prepare the blood smears.The blood smears were stained with Giemsa stain and examined under a light microscope (1000× magnification) for the presence of morulae.An indirect fluorescent-antibody (IFA) method was used for the detection of anti-A.phagocytophilum IgG antibodies employing a commercial IFA kit (Human A. phagocytophila IFA Antibody Kit; Fuller Laboratories, France).Seropositive samples were examined with B. burgdorferi IgG Antibody ELISA kit (Immuno laboratories B. burgdorferi IgG Antibody ELISA, USA) kit to determine the presence of B. burgdorferi IgG antibodies.The tests were performed in the usual manner according to the manufacturer's instructions.

Statistical analysis
Statistical analysis of the published data were performed using Pearson's Chi-square and Fisher's exact tests.The necessary permissions were obtained from the establishment, and informed consent was obtained from each volunteer before participation in the study.

RESULTS
There were no significant differences between gender or age categories for rates of seroprevalance.As shown in Table 1, one sample (0.7%) (67-year-old male) in the IFAT analysis was positive for A. phagocytophilum IgG antibody.When the surveys were checked, this person had a tick bite history three years ago. A. phagocytophilum and B. burgdorferi co-seroprevalence was not observed (Table 1).

DISCUSSION
Around the world in recent years, the use of more effective surveillance programs, the development of new tests for diagnosis, increasing medical importance and tick-borne infections due to environmental changes have increasingly occurred.Additionally, an increase in the number of tick-borne infection cases has been observed.Ticks are one of the most important vectors that transmit many diseases to human and animals worldwide.Although many studies regarding anaplasmosis have been performed worldwide, the number of these studies are increasing since the 2000s (Stuen, 2003;Gokce et al., 2013;Altay et al., 2014;Aktas et al. 2010;Sen et al., 2011).In these studies, the presence of Anaplasma was determined from samples taken from both animals and ticks by serological and molecular methods.Depending on a region's geographic structure, climate and vegetation characteristics, the seroprevalence of tick bites among people varied between 8-25% (Gunes et al., 2011;Kılıc et al., 2010;Ongut et al., 2006).In another study performed on people with a tick bite history, the seroprevalence of A. phagocytophilum was 8%, and the I. ricinus tick population was extremely high, at 80.4% (Ongut et al., 2006).In the Thrace region of Turkey, which is a neighbouring area that has similar environmental conditions to that of Bursa, A. phagocytophilum infection rate was 17.5% in I. ricinus adult ticks in 2008 (Sen et al., 2011).In another study, Göral et al. (1997) found that the seroprevalence of A. phagocytophilum and B. burgdorferi was 35.8% in the forest villages where the I. ricinus tick population was extremely high In all geographical regions of Turkey, more than 30 tick species have been identified in six genera (Rhipicephalus, Dermacentor, Hyalomma, Haemaphysalis, Argas and Ornithodorus) in cattle, sheep and goats (Dumanli, 2012).Because the locations chosen for the study represented the province's meteorological aspect, most of the population makes a living from agriculture and forestry.The high seroprevalence of B. burgdorferi, which is transmitted by the same vector ticks, and the rate of another tick-borne infection, tularaemia, which is found at the highest levels in this region, suggest that Anaplasma can affect people in the Bursa region (Helvacı et al., 2000).In this study, we aimed to determine the seropositivity of Anaplasma infections, not considering whether the participants have tick bite histories, and to determine the risk of Anaplasma infections.According to the 0.7% seropositivity, it was discovered that the inexistence of acute anaplasmosis reports in our region and country, lacked some levels of diagnosis in some cases.Only approximately 24% of the volunteers actually reported a tick bite; however, we noted that the serologically positive sample originated from a volunteer had a tick bite.The HGA infection level has risen since 1999 when HGA was added to the USA list of diseases that should be considered.In 2008, this level was 21% higher than in 2007 (Hall-Baker et al., 2010).The average HGA seroprevalence in Europe is 6.2%, and this level is as high as 28%, in some countries (Bakken and Dumler, 1994).A. phagocytophilum has an enzootic cycle that includes Ixodes ticks and mammalian animals as reservoirs.HGA is transmitted by Ixodes scapularis in the eastern USA and by Ixodes pacificus in the northern USA.In Asia, the Ural Mountains, Siberia, the Far East and Baltic Russia, HGA is transmitted by Ixodes persulcatus (Woldehiwet, 2010).The vector in Europe is I. ricinus (Petrovec et al., 2002;Wittesjö et al., 2001).(Kılıc et al., 2010).The success in finding morulae varies in accordance with the experience of the microscopist and with the duration of the illness (Bakken et al., 2002).Morulae have been detected in only three patients in Europe (Koebel et al., 2012).In this study, morulae were not visualised in the peripheral smear.Because they are transmitted by the same vector (I.ricinus) and have the same epidemiologic features, A. phagocytophilum and B. burgdorferi co-infections are often encountered in humans and animals (Koebel et al., 2012).Güneş and his colleagues reported that the coprevalence rates were 3.3 and 0.55% with respect to B. burgdorferi in two different climatic regions, that is, Sinop and Tokat, respectively (Gunes et al., 2011).In our study, the co-prevalence rate was 0%.This could be attributed to unnoticed tick bites, e.g., bites from nymphs, which are small and feed for relatively shorter times than adults.
Although infestations with ticks such as Hyalomma spp.and Rhipicephalus spp.are recorded as tick bites, these infestations can hardly be considered a source of HGE infection.

Conclusion
In Turkey, there are a limited number of human studies that have focused on this interaction.There is a need for a large-scale population studies especially on tickborne HGA, Rickettsial and Lyme diseases.Future studies on tick-borne diseases should focus more on aspects related to vector and this will be an important component in the study of epidemiology.
+90 224 295 03 22. Fax: +90 224 442 91 81.Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License primary vectors of this parasite.Previously, the A. phagocytophilum parasite was known as the species Ehrlichia phagocytophila, Ehrlichia equi and Ehrlichia platys.This parasite has recently been moved into the genus . Ixodes spp.ticks are *Corresponding author.E-mail: oktayalver@uludag.edu.tr.Tel:

Table 1 .
Demographics and seroprevalence of A. phagocytophilum in persons living in rural areas of Bursa province, Turkey.
Christova et al.found ehrlichiosis agents in Hyalomma, Rhipicephalus and Dermacentor ticks collected from the cities of Antalya, Kayseri and Malatya, Turkey by using PCR and reverse line blot techniques.