MOLECULAR DETECTION OF BABESIA SPP . IN TICKS IN NORTHERN SERBIA

In order to evaluate the prevalence rate of Babesia spp. in ticks collected from vegetation at seven localities in northern Serbia, tick samples were subjected to molecular analysis. A total of 132 unfed adult ticks of five different species (Dermacentor marginatus, Dermacentor reticulatus, Ixodes ricinus, Haemaphysalis concinna and Haemaphysalis punctata), were examined by PCR for the presence of Babesia spp. Out of the analyzed ticks, 10.61% (14/132) were positive for babesial DNA. The presence of babesiae was found at the localities Pančevački Rit, Titov Gaj, Makiš, PKB and Kljajićevo. Prevalence in D. reticulatus ticks was 21.57% (11/51) and in H. concinna ticks, 8.57% (3/35). Sequencing and phylogenetic analysis showed a clustering of the obtained sequences with those of B. canis from the GenBank database. These results add to the knowledge of the distribution of babesial pathogens and their vectors in Serbia.


INTRODUCTION
Babesial parasites (and those of the closely related genus Theileria) are some of the most ubiquitous and widespread blood parasites in the world, after trypanosomes, and as such have considerable worldwide economic, medical, and veterinary impact (Homer et al., 2000).The babesiae are transmitted by ixodid ticks and are capable of infecting a wide variety of vertebrate hosts as reservoirs in maintaining the enzootic transmission cycle, while in susceptible species they bring about disease (Homer et al., 2000;Etkind et al, 1980;Spielman et al., 1981).
Many babesial species have been detected in domestic animals (Babesia bovis, B. equi, B. bigemina, B. ovis, B. canis, B. felis), while B. microti and B. divergens are the most common human pathogens.The first case of human babesiosis was documented in former Yugoslavia in 1957 (Škrabalo and Deanović, 1957).Babesial infections in humans generally follow regional distributions.Cases in the United States are caused primarily by B. microti, whereas in Europe they are usually caused by B. divergens (Quick et al., 1993;Thomford et al., 1994).Both animal and human babesiosis are recognized as emerging zoonoses.
Transovarial transmission is a characteristic of the Babesia genus and in addition to transstadial transmission, ensures persistence within the tick vector, sometimes over several tick generations (Chauvin et al., 2009).
Several studies, based on PCR detection of babesial DNA have provided data about the prevalence of Babesia spp. in various mammalian hosts.The Babesia EU1 prevalence of up to 30% in roe deer (Capreolus capreolus) in Slovenia indicates suggests that this species is a possible reservoir (Duh et al., 2005).The prevalence of B. microti in rodents in northeastern Poland ranges from 11.90% to 35% (Welc-Falęciak et al., 2008).In Greece, 15% of analyzed goats and sheep were infected with B. ovis (Theodoropoulos et al., 2006).All of the eight clinically suspected dogs in Portugal, found to be infected with B. canis by bloodsmear microscopy, were confirmed positive by PCR (Cardoso et al., 2008).Similarly, in Poland 90.80% of naturally infected dogs were PCR-positive for B. canis canis (Adaszek and Winiarczyk, 2008).Based on clinical, microscopic and molecular investigations, 5.90% of dogs analyzed were determined as being infected with babesiae in Slovenia (Duh et al., 2004).In Croatia, B. canis canis was detected in 69%, B. gibsoni in 21%, B. canis vogeli in 7% and Theileria annae in 3% of babesial positive asymptomatic dogs (Beck et al., 2009).In Hungary, molecular analysis of blood samples from dogs with clinical signs of babesiosis, revealed the presence of Babesia spp.DNA in 88.60% (Földvári et al., 2005).In Romania, 12 of 16 tested dogs, both clinically affected and normal, were positive for Babesia spp.(Ionita et al., 2012).A study in Albania revealed that 23% of clinically healthy dogs were positive for B. canis DNA, while IFAT showed a lower prevalence -13% (Hamel et al., 2009).
Previous studies conducted in Serbia analyzed the presence of B. canis in pet dogs and ticks collected from these dogs, by microscopic method.Babesiae were detected in R. sanguineus, Dermacentor marginatus and D. reticulatus ticks with a prevalence of 66.10%, 18.70% and 46.40%, respectively (Pavlović et al, 2002;Pavlović et al., 2009).
As a step forward, the aim of this study was the molecular detection and characterization of Babesia spp. in unfed adult Ixodid ticks (Acari: Ixodidae) collected from several localities in northern Serbia, in order to provide a better understanding of the epidemiological and epizootiological situation in this part of the country.
A total of 132 adult ticks was chosen as a sample of convenience for further analysis.All ticks were morphologically identified to the species level by using existing standard taxonomic keys (Pomerancev, 1950), and split into different Eppendorf test tubes according to species and collection region.Each tick was washed in 70% alcohol, rinsed in sterile water and dried on sterile filter paper.Consequently, they were triturated individually in 500 µl of PBS, into sterile tubes in a laminar flow hood; a portion of the homogenized ticks in PBS was used to make pools according to species and collection region.Each pool contained 2-11 ticks, in a final volume of 200 µl.All samples were stored at -80 o C until DNA extraction.
All DNA extractions were carried out using the QIAamp DNA Tissue kit (QIAGEN-Germany) according to the manufacturer's recommendations.DNA extracts were stored at -20°C until use in PCR amplification.
The detection of Babesia spp.DNA in the pools was performed by PCR amplification, using BABF and BABR primers targeting a 146 bp portion of the 18S rRNA gene sequence (Theodoropoulos et al., 2006).In the case of a positive pool, DNA from the original homogenates was extracted and samples were tested individually.Isolation of amplified DNA from the gel was performed by DNA Extraction Kit (Fermentas, Vilnus, Lithuania) and purification by QIAquick PCR Purification Spin Kit (Qiagen, Germany).PCR products were directly sequenced (Macrogen Inc., Seoul, South Korea).Sequencing revealed six sequences, which were processed using nucleotide Blast (National Center for Biotechnology Information) (http://www.ncbi.nlm.nih.gov/BLAST) and Chromas v1.49, and were aligned using Clus-talW.Two sequences were submitted at the GenBank database under accession numbers HM629446 and HM629447.
The Lasergene Ver.(Thompson et al., 1994).Phylogenetic distances were calculated using the neighbor-joining method.The phylogenetic tree was drawn using N-J plot software.
Out of six sequences obtained, five showed similarities from 99.10% to 100% (Pančevački Rit, PKB, Titov Gaj and Makiš), while the sequence from Kljajićevo was unique with similarity from 98.20% to 99.10%.Genetic similarity analysis showed clustering of the obtained sequences in this study with those of B. canis, from the GenBank database (Fig. 2).

DISCUSSION
In most European countries, the occurrence of B. canis is in accordance with the geographical distribution of its vector, and it is one of the most impor-tant tick-transmitted infectious diseases of dogs in Europe (Ionita et al., 2012;Lobetti, 1998).Instead of sporadic findings of B. canis in other tick species (Cieniuch et al., 2009), Dermacentor reticulatus ticks are known to be vectors of this pathogen in Europe (Janisch, 1986;Ionita et al., 2012), and our study provides additional data about their potential role as vectors in the transmission cycles of B. canis in northern Serbia.In Serbia, this tick species, previously known as Dermacentor pictus, can be found more often in Vojvodina and the Belgrade area, while their distribution in the rest of the country is sporadic (Petrović, 1979;Milutinović, 1992;Tomanović, 2009).Expansion of this species has been recorded in Hungary and Germany (Földvári et al., 2007).
Relative abundance analysis revealed that the species Dermacentor reticulatus was absolutely dominant (38.64%) among the analyzed ticks in this part of Serbia, followed by H. concinna (26.52%),I. ricinus (21.97%),D. marginatus (11.36%) and H. punctata (1.52%).The presence of B. canis DNA was detected in analyzed ticks from five out of the seven localities, on both agricultural and recreational sites.
The Belgrade area is known as a focus of canine babesiosis (Pavlović et al., 2002).Microscopic analysis of the midgut contents of ticks in this region showed the presence of B. canis in engorged R. sanguineus, D. marginatus and D. reticulatus ticks collected from dogs with clinical signs of babesiosis (Pavlović et al., 2009).The prevalence of B. canis in D. reticulatus ticks obtained in that study was 46.40%, while in our research prevalence was 21.57% for the same tick species.Although molecular methods are more sensitive than microscopic ones, the lower prevalence detected in our study is in harmony with the observation of Földvári et al. (2007) that ticks collected from symptomatic dogs brought to veterinary clinics show higher prevalence than unfed ticks.The prevalence of infected unfed ticks is lower, but represents more accurate data for epidemiology research and disease risk assessment.
However, the prevalence of infected unfed D. reticulatus ticks in our study is significantly higher than prevalences published by Rar et al. (2005a) -3.60% andRar et al. (2005b) -4.20%, both in Siberia, and Kubelová et al. (2011) -2.30-14.70% andDuh et al. (2006) -1%, both in Slovakia.The finding of Babesia ssp. in D. reticulatus ticks poses a bigger threat to animal than to human health, since feeding rarely occurs on humans.Adult D. reticulatus ticks feed on larger wild and domestic mammals, and in Serbia, most often on dogs.This species also transmits agents of babesiosis to cattle, horses and sheep (Milutinović et al., 2012).A higher prevalence of infected ticks correlates to a more elevated disease risk and although the occurrence of dog babesiosis declined in the Belgrade area in the period 1997-2004 (Pavlović et al., 2009), data obtained in this study point out the need for constant population monitoring of this tick species.
Recent findings indicate that D. reticulatus ticks carrying Francisella-like endosymbiont (FLE) may have a higher vector potential for B. canis, but further research is necessary to confirm this (Tomanović et al., in press).
The Kljajićevo locality was the only one where babesial DNA-positive H. concinna ticks were found.To our knowledge, this was a unique finding of the presence of B. canis in this species.This and other data about the presence of various pathogens in this tick indicate its potential epidemiological and epizootiological significance in this area (Tomanović ).Adult ticks of this species usually feed on large and medium large mammals, wild or domestic.Adults and nymphs can also be found on humans (Milutinović et al., 2012).The possible role of H. concinna ticks in maintenance of the enzootic cycle of B. canis requires further research.
Six different 146 bp-long babesial 18S rDNA sequences were obtained.Sequences from Pančevački Rit, Titov Gaj and one from PKB were identical, as well as sequences from Makiš and the PKB locality.The sequence from Kljajićevo was unique, showing 98.20% and 99.10% similarity with the previous two, respectively.The fact that the sequence from Kljajićevo was obtained from H. concinna as the first detection of Babesia spp. in this tick species, implicates further detailed molecular characterization.
Ixodes ricinus is one of the most widely distributed and abundant tick species in Serbia (Petrović, 1979;Milutinović, 1992;Milutinović and Radulović, 2002).Adults feed on large wild mammals and almost all domestic animals, as well as birds (Milutinović et al., 2012).Nymphs and adults can often be found on humans (Estrada-Peña and Jongejan, 1999;Hubálek et al., 2004).No Babesia DNA-positive I. ricinus ticks were detected during our study, probably due to the small number of analyzed ticks, taking into account the low prevalence rates (0.10-5%) of Babesia spp. in I. ricinus ticks obtained in several studies throughout in Europe (Cieniuch et al., 2009;Reye et al., 2010;Cassini et al., 2010;Welc-Falęciak et al., 2008).Moreover, the presence of Babesia spp. in I. ricinus ticks at these localities could not be excluded.
Our detection of B. canis in ticks from the Kljajićevo locality represents the most northern finding of this pathogen in Serbia.In Hungary, the presence of babesiae in regions where babesiosis have not been reported earlier as been noted (Földvári et al., 2007).In Romania, an increased prevalence of dog babesiosis has been registered in the last years (Ionita et al., 2012).
Investigations of tick fauna and pathogen prevalence rates in ticks as vectors are essential for risk as-sessment and adequate prevention of tick-transmitted diseases.Although Fritz (2009) claimed that no canine Babesia spp.seemed to be zoonotic, cases of human babesiosis in France caused by B. canis (Marsaudon et al., 1995) indicate the possible crossing of species barriers and its zoonotic potential.The molecular approach in the detection of Babesia spp., as a sensitive and specific tool, has been used for the first time in Serbia in this study.The data on the presence of B. canis in D. reticulatus and H. concinna ticks in the Belgrade region and northern Serbia provide better insight into the transmission cycles and maintenance of babesiae in Serbia.Further studies need to be carried out to eliminate knowledge gaps in the ecology, epizootiology, and epidemiology of babesiosis in Serbia.

Fig. 2 .
Fig. 2. Phylogenetic tree based on partial sequences of Babesia 18S rRNA gene obtained in this study and those deposited in GenBank (accession numbers are indicated next to each sequence).Plasmodium falciparum was used as a outgroup.Phylogenetic distances were calculated using the neighbour-joining method.

Table 1 .
Prevalence of Babesia spp. in ticks from northern Serbia