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Original article
Anaplasma phagocytophilum in questing Ixodes ricinus ticks from Romania Ioana Adriana Matei ∗ , Zsuzsa Kalmár, Cristian Magdas¸, Virginia Magdas¸, Hortenzia Toriay, Mirabela Oana Dumitrache, Angela Monica Ionic˘a, Gianluca D’Amico, Attila D. Sándor, Daniel Ioan M˘arcut¸an, Cristian Doms¸a, C˘alin Mircea Gherman, Andrei Daniel Mihalca Faculty of Veterinary Medicine, University of Agricultural Sciences and Veterinary Medicine, Department of Parasitology and Parasitic Diseases, Cluj Napoca, Cluj, Romania
a r t i c l e
i n f o
Article history: Received 12 September 2014 Received in revised form 21 January 2015 Accepted 9 March 2015 Available online xxx Keywords: Anaplasma phagocytophilum Ixodes ricinus Romania
a b s t r a c t Granulocytic anaplasmosis is a common vector-borne disease of humans and animals with natural transmission cycle that involves tick vectors, among which Ixodes ricinus is the most important. The present paper reports the prevalence and geographical distribution of A. phagocytophilum in 10,438 questing Ixodes ricinus ticks collected at 113 locations from 40 counties of Romania. The unfed ticks were examined for the presence of A. phagocytophilum by PCR targeting a portion of ankA gene. The overall prevalence of infection was 3.42%, with local prevalences ranging between 0.29% and 22.45%, with an average prevalence of 5.39% in the infected localities. The infection with A. phagocytophilum was detected in 72 out of 113 localities and in 34 out of 40 counties. The highest prevalence was recorded in females followed by males and nymphs. The results and the distribution model have shown a large distribution of A. phagocytophilum, covering Romania’s entire territory. This study is the first large scale survey of the presence of A. phagocytophilum in questing I. ricinus ticks from Romania. © 2015 Elsevier GmbH. All rights reserved.
Introduction Anaplasma phagocytophilum, is a small, pleomorphic, Gramnegative, obligate intracellular bacterial organism that typically infects granulocytes of mammals (Chen et al., 1994; Dumler et al., 2001). The disease is known as tick-borne fever (TBF) in ruminants, human granulocytic anaplasmosis (HGA) in people, canine granulocytic anaplasmosis (CGA) in dogs and equine granulocytic anaplasmosis (EGA) in horses (Dumler et al., 2001). Infections with A. phagocytophilum were found in animals and humans from most parts of the northern hemisphere. It was detected in North America (USA and Canada) and in almost all countries of Europe (Strle, 2004; Teglas and Foley, 2006). From Asia, it was detected in Turkey, Russia, China, Korea and Japan (Aktas et al., 2011; Cao et al., 2000; Ohashi et al., 2005; Kim et al., 2006). Reports of A. phagocytophilum or closely related strains from Africa and South America are occasional (Inokuma et al., 2005; André et al.,
∗ Corresponding author at: Calea Manastur 3-5, Cluj Napoca, Cluj, Romania. E-mail address: [email protected] (I.A. Matei).
2012, 2014). There is no reliable data available about its occurrence in South America and Australia. Ticks of the genus Ixodes are the main vectors for A. phagocytophilum (Stuen et al., 2013). In Europe, the most important vector is Ixodes ricinus (Strle, 2004). This tick is widespread and has a very broad host spectrum, being also the dominant tick in Romania, including on humans (Briciu et al., 2011; Mihalca et al., 2012b,c). Despite the common presence of A. phagocytophilum in Europe, its first molecular identification in Romania was in 2012, when P˘aduraru et al. (2012) identified it in I. ricinus collected from roe deer (Capreolus capreolus) and goats in 2 sites in eastern Romania. Subsequently, several other reports on its presence or prevalence were published in dogs (Hamel et al., 2012; Mircean et al., 2012) and wild boars (Sus scrofa) (Kiss et al., 2014), as well as in ticks collected from livestock (Ionit¸a˘ et al., 2013), hedgehogs (Erinaceus roumanicus) (Dumitrache et al., 2013), tortoises (Testudo graeca ibera) (Pas¸tiu et al., 2012) and birds (M˘arcut¸an et al., 2014). Despite the climatic and habitat heterogeneity of Romania (Donit¸a˘ et al., 2005), the prevalence and distribution of A. phagocytophilum in questing ticks has never been evaluated so far. Although I. ricinus has a wide distribution across Romania (Mihalca et al., 2012c), the reservoir hosts such as wild ruminants, wild boars, rodents
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Please cite this article in press as: Matei, I.A., et al., Anaplasma phagocytophilum in questing Ixodes ricinus ticks from Romania. Ticks Tick-borne Dis. (2015), http://dx.doi.org/10.1016/j.ttbdis.2015.03.010
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DNA extraction Genomic DNA extraction was performed individually from each tick with ammonium hydroxide (Morán-Cadenas et al., 2007). Ticks were boiled in 300 l of 1.25% ammonium hydroxide at 100 ◦ C for 30 min, and then cooled. The tubes with the lysate were left open for 30 min at 100 ◦ C for the ammonia to evaporate. In order to assess cross-contamination between extracts, negative controls were used in each extraction procedure. The DNA quantity and purity were assessed on Nanodrop ND-1000 spectrophotometer analyzer (NanoDrop Technologies, Inc., Wilmington, DE, USA), using a representative number of randomly selected samples.
Polymerase chain reaction (PCR)
Fig. 1. Prevalence of A. phagocytophilum in questing I. ricinus ticks from Romania.
and insectivores (Stuen et al., 2013) may have a variable density and distribution in Romania (Wagner, 1974, 1976). With this view, the aims of our study were to evaluate the prevalence of A. phagocytophilum in questing I. ricinus ticks from multiple locations distributed throughout Romania and to correlate its presence with different ecological features.
Materials and methods Tick collection and sampling sites The tick sampling took place between March and May 2010 and between March and May 2011, using the dragging method ˜ 2001). A random sampling approach was used, as (Estrada-Pena, previously described (Kalmár et al., 2013). Grid cells of 10 × 10 km were designed to cover the entire country. Only cells having forests were considered appropriate for sampling (Mihalca et al., 2012c). All the grid cells with negligible forest coverage (50% by bootstrap analysis. Evolutionary analyses were conducted using MEGA6 software (Tamura et al., 2013).
Please cite this article in press as: Matei, I.A., et al., Anaplasma phagocytophilum in questing Ixodes ricinus ticks from Romania. Ticks Tick-borne Dis. (2015), http://dx.doi.org/10.1016/j.ttbdis.2015.03.010
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Fig. 2. A. phagocytophilum distribution model in Romania using maximum entropy species distribution approach (Maxent software). Model was created with the following grids: altitude, BIO1 – annual mean temperature and BIO12 – annual precipitation. Green indicating conditions typical of those where the species is found, and lighter shades of blue indicating low predicted probability of suitable conditions. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) Fig. 3. Phylogenetic analysis based on ankA gene of Anaplasma phagocytophilum.
Accession numbers GenBank nucleotide accession numbers of ankA gene sequences from this study are: KP164412, KP164413, KP164414, and KP164415. Statistical analysis and GIS modeling Statistical calculations were performed using Epi InfoTM 7 (CDC, USA) software and analyzed by chi-squared independence: the total infection prevalence of A. phagocytophilum and the 95% confidence interval, infection prevalence differentiated by developmental stages and sex of I. ricinus and the infection prevalence in each location. The probability of prevalence higher than 1% was calculated according to altitude, medium annual temperature and annual precipitation levels. The altitude and climatic data were obtained from www.worldclim.org (Altitude, BIO1 – annual mean temperature and BIO12 – annual precipitation). In order to design the distribution model, we considered only positive locations. The model was designed using Maxent software by employing maximum entropy species distribution approach. The grids used for the model were altitude, BIO1 – annual mean temperature and BIO12 – annual precipitation. Statistical significance of the prediction (sensivity vs. specificity) and the contribution of each variable were calculated. Maps with location points and with A. phagocytophilum prevalence degree were generated using ArcGis/ArcMap software. Results A total of 4655 nymphs (44.41%), 2943 females (28.07%) and 2885 males (28.52%) (total n = 10,483) of questing I. ricinus ticks were analyzed for the presence of A. phagocytophilum using PCR. The BLAST analysis of all randomly selected sequences showed a 99% similarity with A. phagocytophilum strain Bel11-2-07 ankryin (accession number HQ629927) isolated from Estonian I. ricinus and 98% similarity with an A. phagocytophilum isolate red deer 473 (accession number GU236718) isolated from Cervus elaphus in Slovenia. Phylogenetic tree (Fig. 3) analysis reveals the two major clusters of ankA gene described before by Scharf et al. (2011),
which contain strains isolated from humans, dogs, horses, cats and ticks and from roe deer and tick samples, respectively. Our sequences A. phagocytophilum RO1 (KP164415), RO2 (KP164412), and RO4 (KP164414), respectively, are highly similar with A. phagocytophilum obtained from wild boar from Poland and with other strains of A. phagocytophilum isolated from I. ricinus. A. phagocytophilum RO3 (KP164413) forms a paraphyletic group with A. phagocytophilum isolated from different animal tissues, particularly from roe deer (Fig. 3). The overall prevalence of infection was 3.42% (359/10,483; 95% CI: 3.09–3.80). The highest prevalence was recorded in adult females (8.7%; 256/2943; 95% CI: 7.72–7.79), followed by adult males (2.46%; 71/2885; 95% CI: 1.94–3.11) and nymphs (0.69%; 32/4655; 95% CI: 0.48–0.98) (x2 = 361.1, df = 2, p < 0.00001). The overall prevalence in adult ticks (5.56%; 327/5883; 95% CI: 4.99–6.18) was higher than that in nymphs (0.69%; 32/4655; 95% CI: 0.48–0.98) (x2 = 184.5, df = 1, p < 0.00001). The infection with A. phagocytophilum was detected in 72 out of 113 localities (63.72%; 95% CI: 54.14–72.55) and in 34 out of 40 counties (85.00%; 95% CI: 70.16–94.29). The local prevalence in the positive localities varied between 0.29% and 22.45%, with an average value of 5.39% (359/6658; 95% CI: 4.87–5.97). The local prevalence for the positive collection sites is shown in Fig. 1. A prevalence higher than 1% was more common at altitudes below 350 m (x2 = 5.21, df = 1, p < 0.05) and at average annual temperatures higher than 9.5 ◦ C (x2 = 4.3, df = 1, p < 0.05). The distribution model (Fig. 2) has shown a medium prediction (AUC = 0.614) (Fig. S1 in Supplementary file). The altitude had the highest influence on the distribution (Fig. S2 in Supplementary file), the presence of A. phagocytophilum being negatively associated with the altitude (Supplementary file S2). Discussion Only few scattered data concerning the epidemiology of A. phagocytophilum in Romania are available. The present study is the first survey on the presence of A. phagocytophilum in questing ticks from this country. In I. ricinus ticks collected from
Please cite this article in press as: Matei, I.A., et al., Anaplasma phagocytophilum in questing Ixodes ricinus ticks from Romania. Ticks Tick-borne Dis. (2015), http://dx.doi.org/10.1016/j.ttbdis.2015.03.010
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domestic hosts in three counties from the southern part and one from the northern part of the country, the prevalence of A. phagocytophilum was 6.7% (Ionit¸a˘ et al., 2013). A study on the prevalence of A. phagocytophilum in I. ricinus collected from roe deer and goats in two counties from southern Romania found a prevalence of 1.4% (P˘aduraru et al., 2012). Another study was performed on I. ricinus collected from hedgehogs (Erinaceus roumanicus) by Dumitrache et al. (2013), where the authors found 12% prevalence. Neither of these studies evaluated the seasonal variation. Moreover, no evaluation on the host infection was performed. Interestingly, Pas¸tiu et al. (2012) found a relatively high prevalence (18.8%) of A. phagocytophilum in Hyalomma aegyptium ticks collected from tortoises (Testudo graeca) in southeastern Romania, suggesting a rather passive carrier role of these ticks but also an epidemiological proof for their host switching behavior (Pas¸tiu et al., 2012). Additionally, a recent study from western Romania has provided evidence for the role of wild boars (Sus scrofa) as potentially important reservoir hosts for A. phagocytophilum (Kiss et al., 2014). No data on human clinical cases have been published so far in Romania. A. phagocytophilum has been detected in ticks from all over the world, mostly in the northern hemisphere (Strle, 2004; Teglas and Foley, 2006). In Europe it was recorded from almost all countries with prevalence ranging between 0.5% and 34% (Christova et al., 2001; Egyed et al., 2012). The overall prevalence we recorded falls within the European average (Strle, 2004). However, the nationwide A. phagocytophilum prevalence may be lower since the locations taken in our study had considerable forest coverage and previous studies have shown a higher prevalence in this type of habitat (Halos et al., 2006, 2010). In each country and between countries the prevalence seems to be variable. In Central and Eastern Europe, high prevalence variability was found in the different countries, from low values in Hungary and Republic of Moldova (0.5–2.4%), medium values in Slovakia and Russia (8–9%) to high values of 34% in Bulgaria (Christova et al., 2001; Masuzawa et al., 2008; Egyed et al., 2012; Subramanian et al., 2012; Movila et al., 2013). Several factors were suggested to explain these local variations in prevalence, the most commonly incriminated being the abundance and population structure of the tick vector and the abundance and diversity of potential reservoir hosts. The population ecology of the vector ticks depends on complex factors, such as the type of habitat, climatic conditions (related mostly to latitude and altitude) but also the host availability (Gern and Humair, 2002). Population structure and abundance of the vector depend also on the seasonal activity, I. ricinus being more active in the spring (Gern and Humair, 2002); hence, the ratio of adults and nymphs will influence the overall prevalence. More studies have shown differences in the prevalence of A. phagocytophilum between developmental stages, which is higher in adults than in nymphs. Our results have also shown a higher prevalence of A. phagocytophilum in adults compared to nymphs (5.56% vs. 0.69%). Similar results were recorded in other European countries (Christova et al., 2001; Overzier et al., 2013; Venclikova et al., 2014; Granquist et al., 2014). These differences in A. phagocytophilum prevalence could be explained by the higher probability of the adults to feed on an infected reservoir host, especially since transovarial transmission of A. phagocytophilum has not yet been demonstrated. Among adult ticks, females seem to be more often infected with A. phagocytophilum (Rosef et al., 2009). Although the sampling in the current study was done exclusively during the spring, previous studies have shown little or no significant seasonal variation in the prevalence of A. phagocytophilum (Silaghi et al., 2008). However, others found contradicting results: higher prevalence in spring (Mysterud et al., 2013) or higher prevalence during autumn (Reye et al., 2010). Geographical patterns and habitat structure were demonstrated as important factors not only for the abundance of ticks, but also for
the prevalence of A. phagocytophilum infection in vectors. Various studies reported a focal distribution of the infection, with significant differences between urban parks, pasture and forest (Ogden et al., 1998; Wielinga et al., 2006; Halos et al., 2006, 2010). The prevalence in ticks may also be influenced by the A. phagocytophilum epidemiology in the reservoir hosts, given that different developmental stages of ticks may have different preferred host species (Feider, 1965; Mihalca et al., 2012b). In an extensive study regarding the distribution of ticks and host associations in Romania, some patterns were observed for Ixodes ricinus (Mihalca et al., 2012a,b). For instance, on the majority of rodent species, exclusively immature stages of I. ricinus were observed. Adult I. ricinus were found only on three rodent species (Apodemus agrarius, Microtus arvalis and Micromys minutus). On insectivores, wild ruminants and humans, all stages were observed, while on wild carnivores, only nymphs and adults were found. Wild boars (Sus scrofa) were infested only with adults (Mihalca et al., 2012b). Similar observations were made for birds, adults being found only in few instances (Mihalca et al., 2012b; Sándor et al., 2014). An interesting aspect was the correlation with the average altitude, with a higher presence of A. phagocytophilum in localities below 350 m altitude (x2 = 5.21, df = 1, p < 0.03). This result is similar with previous observations, according to which A. phagocytophilum prevalence is negatively correlated with altitude. In the study of Grøva et al. (2011) a smaller prevalence of A. phagocytophilum infection was observed in lambs grazing at high altitude compared with farms grazing at low altitude. Additionally Gilbert (2010) found that the density of I. ricinus is decreasing at increasing altitudes. Furthermore, we found a positive correlation with the local average annual temperature. Higher prevalence was found at an average temperature exceeding 9.5 ◦ C (x2 = 4.3, df = 1, p < 0.04). Influence of the altitude and temperature on the prevalence of A. phagocytophilum could be explained by the lower vector abundance (Gilbert, 2010). Differences in reservoir hosts communities could also influence the prevalence of A. phagocytophilum in questing ticks from the same habitats. Gilbert (2010) tested the effect of different hosts on tick abundance depending on the altitude. Her models have shown an increasing abundance of larvae and nymphs with the increasing deer abundance. The effect of deer abundance on nymphs was significant at lower altitudes and not significant at high altitudes. Moreover, a negative correlation was observed between the deer abundance and altitude. For the mountain hare abundance, a negative trend on larval abundance was observed, as the relation between mountain hare and altitude was positive (Gilbert, 2010). Similarly, the structure of rodent and bird communities, which are potential reservoir and dispersal hosts for A. phagocytophilum (Rejmanek et al., 2011; Capligina et al., 2014; Lommano et al., 2014) may also differ at various altitudes and temperatures (Wagner, 1974, 1976). In a previous study, the ankA gene analysis has shown a correlation between host species and the four identified A. phagocytophilum ankA gene clusters (Scharf et al., 2011). Sequences from dogs, humans, horses and cats were found exclusively in cluster I. Overall, sequences from ruminants were diverse and were found in all clusters, but particularly, sequences from roe deer were found exclusively in clusters II and III, while other ruminants (e.g. red deer, sheep, bison, cows) were mainly in clusters I and IV (Scharf et al., 2011). In the present study, phylogenetic tree analysis has shown two genetic lineages. One contains the sequences RO1 (KP164415), RO2 (KP164412) and RO4 (KP164414) and sequences isolated from ticks and animal tissues which were previously classified in cluster I. The second one includes the sequence RO3 (KP164413) and sequences isolated from ruminant tissues, mainly roe deer. These results suggest a large variety of host species for A. phagocytophilum in Romania. Wild boars as host for A. phagocytophilum were previously studied (Kiss et al., 2014), but further analysis regarding wild
Please cite this article in press as: Matei, I.A., et al., Anaplasma phagocytophilum in questing Ixodes ricinus ticks from Romania. Ticks Tick-borne Dis. (2015), http://dx.doi.org/10.1016/j.ttbdis.2015.03.010
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ruminants, domestic carnivores and horses are required in order to understand the epidemiology of A. phagocytophilum in Romania. According to the distribution model, most of Romania’s territory has typical conditions for the presence of A. phagocytophilum, but the risk is generally moderate. The risk of acquiring A. phagocytophilum associated with I. ricinus bite is lower in mountainous areas, as it is negatively correlated with altitude. Acknowledgments ADM, ADS, CD and GD were supported in this research by a grant from the UEFISCDI grant PCE 236/2011. CM was supported by a grant from USAMV 1215/28/2012. CD, ZK, DIM and MOD’s work was financed by POSDRU grant no. 159/1.5/S/136893 grant with title: “Parteneriat strategic pentru cres¸terea calit˘at¸ii cercet˘arii s¸tiint¸ifice din universit˘at¸ile medicale prin acordarea de burse doctorale s¸i postdoctorale – DocMed.Net 2.0”. ADM, MOD, ZK, IAM, AMI, GD and ADS are members of the COST Action TD1303 “European Network for Neglected Vectors and Vector-Borne Infections (EURNEGVEC)”. We would like to thank our colleagues Mircean Viorica, Adriana Györke, Ioana Pas¸tiu and Miruna Oltean for their help during the field work. Special thanks to Friederike von Loewenich for all her valuable advices during the preparation of this manuscript. This paper was published under the frame of EurNegVec COST Action TD1303. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.ttbdis.2015.03.010. References Aktas, M., Altay, K., Dumanli, N., 2011. Molecular detection and identification of Anaplasma and Ehrlichia species in cattle from Turkey. Ticks Tick Borne Dis. 2, 62–65. André, M.R., Baccarim Denardi, N.C., Marques de Sousa, K.C., Gonc¸alves, L.R., Henrique, P.C., Grosse Rossi Ontivero, C.R., Lima Gonzalez, I.H., Vaz Cabral Nery, C., Romeiro Fernandes Chagas, C., Monticelli, C., Alexandre de Santis, A.C.G., Machado, R.Z., 2014. Arthropod-borne pathogens circulating in freeroaming domestic cats in a zoo environment in Brazil. Ticks Tick Borne Dis., http://dx.doi.org/10.1016/j.ttbdis.2014.03.011. André, M.R., Dumler, J.S., Scorpio, D.G., Teixeira, R.H.F., Allegretti, S.M., Machado, R.Z., 2012. Molecular detection of tick-borne bacterial agents in Brazilian and exotic captive carnivores. Ticks Tick Borne Dis. 3, 247–253. Briciu, V.T., Titilincu, A., T˘at¸ulescu, D.F., Carstina, D., Lefkaditis, M., Mihalca, A.D., 2011. First survey on hard ticks Ixodidae collected from humans in Romania: possible risks for tick-borne diseases. Exp. Appl. Acarol. 54, 199–204. Cao, W.C., Zhoa, Q.M., Zhang, P.H., Dumler, J.S., Zhang, X.T., Fang, L.Q., Yang, H., 2000. Granulocytic Ehrlichiae in Ixodes persulcatus ticks from an area in China where lyme disease is endemic. J. Clin. Microbiol. 38, 4208–4210. Capligina, V., Salmane, I., Keiˇss, O., Vilks, K., Japina, K., Baumanis, V., Ranka, R., 2014. Prevalence of tick-borne pathogens in ticks collected from migratory birds in Latvia. Ticks Tick Borne Dis. 5, 75–81. Caturegli, P., Asanovich, K.M., Walls, J.J., Bakken, J.S., Madigan, J.E., Popov, V.L., Dumler, J.S., 2000. AnkA: an Ehrlichia phagocytophila group gene encoding a cytoplasmic protein antigen with ankyrin repeats. Infect. Immun. 68, 5277–5283. Chen, S.M., Dumler, J.S., Bakken, J.S., Walker, D.H., 1994. Identification of a granulocytotropic Ehrlichia species as the etiologic agent of human disease. J. Clin. Microbiol. 32, 589–595. Christova, I., Schouls, L., van de Pol, I., Park, J., Panayotov, S., Lefterova, V., Kantardjiev, T., Dumler, J.S., 2001. High prevalence of granulocytic Ehrlichiae and Borrelia burgdorferi sensu lato in Ixodes ricinus ticks from Bulgaria. J. Clin. Microbiol. 39, 4172–4174. Donit¸a˘ , N., Popescu, A., Pauc˘a-Com˘anescu, M., Mih˘ailescu, S., Biris¸, I.A., 2005. Habitats from Romania. Tehnica Silvica, Bucharest, pp. 377–384 (in Romanian). Dumitrache, M.O., Pas¸tiu, A.I., Kalmár, Z., Mircean, V., Sándor, A.D., Gherman, C.M., Cozma, V., 2013. Northern white-breasted hedgehogs Erinaceus roumanicus as hosts for ticks infected with Borrelia burgdorferi sensu lato and Anaplasma phagocytophilum in Romania. Ticks Tick Borne Dis. 4, 214–217. Dumler, J.S., Barbet, A.F., Bekker, C.P.J., Dasch, G.A., Palmer, G.H., Ray, S.C., Rikihisa, Y., Rurangirwa, F.R., 2001. Reorganization of genera in the families Rickettsiaceae and Anaplasmataceae in the order Rickettsiales: unification of some species of Ehrlichia with Anaplasma. Cowdria with Ehrlichia, and Ehrlichia with Neorickettsia, descriptions of six new species combinations and designation of Ehrlichia
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Original article
Anaplasma phagocytophilum in questing Ixodes ricinus ticks from Romania Ioana Adriana Matei ∗ , Zsuzsa Kalmár, Cristian Magdas¸, Virginia Magdas¸, Hortenzia Toriay, Mirabela Oana Dumitrache, Angela Monica Ionic˘a, Gianluca D’Amico, Attila D. Sándor, Daniel Ioan M˘arcut¸an, Cristian Doms¸a, C˘alin Mircea Gherman, Andrei Daniel Mihalca Faculty of Veterinary Medicine, University of Agricultural Sciences and Veterinary Medicine, Department of Parasitology and Parasitic Diseases, Cluj Napoca, Cluj, Romania
a r t i c l e
i n f o
Article history: Received 12 September 2014 Received in revised form 21 January 2015 Accepted 9 March 2015 Available online xxx Keywords: Anaplasma phagocytophilum Ixodes ricinus Romania
a b s t r a c t Granulocytic anaplasmosis is a common vector-borne disease of humans and animals with natural transmission cycle that involves tick vectors, among which Ixodes ricinus is the most important. The present paper reports the prevalence and geographical distribution of A. phagocytophilum in 10,438 questing Ixodes ricinus ticks collected at 113 locations from 40 counties of Romania. The unfed ticks were examined for the presence of A. phagocytophilum by PCR targeting a portion of ankA gene. The overall prevalence of infection was 3.42%, with local prevalences ranging between 0.29% and 22.45%, with an average prevalence of 5.39% in the infected localities. The infection with A. phagocytophilum was detected in 72 out of 113 localities and in 34 out of 40 counties. The highest prevalence was recorded in females followed by males and nymphs. The results and the distribution model have shown a large distribution of A. phagocytophilum, covering Romania’s entire territory. This study is the first large scale survey of the presence of A. phagocytophilum in questing I. ricinus ticks from Romania. © 2015 Elsevier GmbH. All rights reserved.
Introduction Anaplasma phagocytophilum, is a small, pleomorphic, Gramnegative, obligate intracellular bacterial organism that typically infects granulocytes of mammals (Chen et al., 1994; Dumler et al., 2001). The disease is known as tick-borne fever (TBF) in ruminants, human granulocytic anaplasmosis (HGA) in people, canine granulocytic anaplasmosis (CGA) in dogs and equine granulocytic anaplasmosis (EGA) in horses (Dumler et al., 2001). Infections with A. phagocytophilum were found in animals and humans from most parts of the northern hemisphere. It was detected in North America (USA and Canada) and in almost all countries of Europe (Strle, 2004; Teglas and Foley, 2006). From Asia, it was detected in Turkey, Russia, China, Korea and Japan (Aktas et al., 2011; Cao et al., 2000; Ohashi et al., 2005; Kim et al., 2006). Reports of A. phagocytophilum or closely related strains from Africa and South America are occasional (Inokuma et al., 2005; André et al.,
∗ Corresponding author at: Calea Manastur 3-5, Cluj Napoca, Cluj, Romania. E-mail address: [email protected] (I.A. Matei).
2012, 2014). There is no reliable data available about its occurrence in South America and Australia. Ticks of the genus Ixodes are the main vectors for A. phagocytophilum (Stuen et al., 2013). In Europe, the most important vector is Ixodes ricinus (Strle, 2004). This tick is widespread and has a very broad host spectrum, being also the dominant tick in Romania, including on humans (Briciu et al., 2011; Mihalca et al., 2012b,c). Despite the common presence of A. phagocytophilum in Europe, its first molecular identification in Romania was in 2012, when P˘aduraru et al. (2012) identified it in I. ricinus collected from roe deer (Capreolus capreolus) and goats in 2 sites in eastern Romania. Subsequently, several other reports on its presence or prevalence were published in dogs (Hamel et al., 2012; Mircean et al., 2012) and wild boars (Sus scrofa) (Kiss et al., 2014), as well as in ticks collected from livestock (Ionit¸a˘ et al., 2013), hedgehogs (Erinaceus roumanicus) (Dumitrache et al., 2013), tortoises (Testudo graeca ibera) (Pas¸tiu et al., 2012) and birds (M˘arcut¸an et al., 2014). Despite the climatic and habitat heterogeneity of Romania (Donit¸a˘ et al., 2005), the prevalence and distribution of A. phagocytophilum in questing ticks has never been evaluated so far. Although I. ricinus has a wide distribution across Romania (Mihalca et al., 2012c), the reservoir hosts such as wild ruminants, wild boars, rodents
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Please cite this article in press as: Matei, I.A., et al., Anaplasma phagocytophilum in questing Ixodes ricinus ticks from Romania. Ticks Tick-borne Dis. (2015), http://dx.doi.org/10.1016/j.ttbdis.2015.03.010
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DNA extraction Genomic DNA extraction was performed individually from each tick with ammonium hydroxide (Morán-Cadenas et al., 2007). Ticks were boiled in 300 l of 1.25% ammonium hydroxide at 100 ◦ C for 30 min, and then cooled. The tubes with the lysate were left open for 30 min at 100 ◦ C for the ammonia to evaporate. In order to assess cross-contamination between extracts, negative controls were used in each extraction procedure. The DNA quantity and purity were assessed on Nanodrop ND-1000 spectrophotometer analyzer (NanoDrop Technologies, Inc., Wilmington, DE, USA), using a representative number of randomly selected samples.
Polymerase chain reaction (PCR)
Fig. 1. Prevalence of A. phagocytophilum in questing I. ricinus ticks from Romania.
and insectivores (Stuen et al., 2013) may have a variable density and distribution in Romania (Wagner, 1974, 1976). With this view, the aims of our study were to evaluate the prevalence of A. phagocytophilum in questing I. ricinus ticks from multiple locations distributed throughout Romania and to correlate its presence with different ecological features.
Materials and methods Tick collection and sampling sites The tick sampling took place between March and May 2010 and between March and May 2011, using the dragging method ˜ 2001). A random sampling approach was used, as (Estrada-Pena, previously described (Kalmár et al., 2013). Grid cells of 10 × 10 km were designed to cover the entire country. Only cells having forests were considered appropriate for sampling (Mihalca et al., 2012c). All the grid cells with negligible forest coverage (50% by bootstrap analysis. Evolutionary analyses were conducted using MEGA6 software (Tamura et al., 2013).
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Fig. 2. A. phagocytophilum distribution model in Romania using maximum entropy species distribution approach (Maxent software). Model was created with the following grids: altitude, BIO1 – annual mean temperature and BIO12 – annual precipitation. Green indicating conditions typical of those where the species is found, and lighter shades of blue indicating low predicted probability of suitable conditions. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) Fig. 3. Phylogenetic analysis based on ankA gene of Anaplasma phagocytophilum.
Accession numbers GenBank nucleotide accession numbers of ankA gene sequences from this study are: KP164412, KP164413, KP164414, and KP164415. Statistical analysis and GIS modeling Statistical calculations were performed using Epi InfoTM 7 (CDC, USA) software and analyzed by chi-squared independence: the total infection prevalence of A. phagocytophilum and the 95% confidence interval, infection prevalence differentiated by developmental stages and sex of I. ricinus and the infection prevalence in each location. The probability of prevalence higher than 1% was calculated according to altitude, medium annual temperature and annual precipitation levels. The altitude and climatic data were obtained from www.worldclim.org (Altitude, BIO1 – annual mean temperature and BIO12 – annual precipitation). In order to design the distribution model, we considered only positive locations. The model was designed using Maxent software by employing maximum entropy species distribution approach. The grids used for the model were altitude, BIO1 – annual mean temperature and BIO12 – annual precipitation. Statistical significance of the prediction (sensivity vs. specificity) and the contribution of each variable were calculated. Maps with location points and with A. phagocytophilum prevalence degree were generated using ArcGis/ArcMap software. Results A total of 4655 nymphs (44.41%), 2943 females (28.07%) and 2885 males (28.52%) (total n = 10,483) of questing I. ricinus ticks were analyzed for the presence of A. phagocytophilum using PCR. The BLAST analysis of all randomly selected sequences showed a 99% similarity with A. phagocytophilum strain Bel11-2-07 ankryin (accession number HQ629927) isolated from Estonian I. ricinus and 98% similarity with an A. phagocytophilum isolate red deer 473 (accession number GU236718) isolated from Cervus elaphus in Slovenia. Phylogenetic tree (Fig. 3) analysis reveals the two major clusters of ankA gene described before by Scharf et al. (2011),
which contain strains isolated from humans, dogs, horses, cats and ticks and from roe deer and tick samples, respectively. Our sequences A. phagocytophilum RO1 (KP164415), RO2 (KP164412), and RO4 (KP164414), respectively, are highly similar with A. phagocytophilum obtained from wild boar from Poland and with other strains of A. phagocytophilum isolated from I. ricinus. A. phagocytophilum RO3 (KP164413) forms a paraphyletic group with A. phagocytophilum isolated from different animal tissues, particularly from roe deer (Fig. 3). The overall prevalence of infection was 3.42% (359/10,483; 95% CI: 3.09–3.80). The highest prevalence was recorded in adult females (8.7%; 256/2943; 95% CI: 7.72–7.79), followed by adult males (2.46%; 71/2885; 95% CI: 1.94–3.11) and nymphs (0.69%; 32/4655; 95% CI: 0.48–0.98) (x2 = 361.1, df = 2, p < 0.00001). The overall prevalence in adult ticks (5.56%; 327/5883; 95% CI: 4.99–6.18) was higher than that in nymphs (0.69%; 32/4655; 95% CI: 0.48–0.98) (x2 = 184.5, df = 1, p < 0.00001). The infection with A. phagocytophilum was detected in 72 out of 113 localities (63.72%; 95% CI: 54.14–72.55) and in 34 out of 40 counties (85.00%; 95% CI: 70.16–94.29). The local prevalence in the positive localities varied between 0.29% and 22.45%, with an average value of 5.39% (359/6658; 95% CI: 4.87–5.97). The local prevalence for the positive collection sites is shown in Fig. 1. A prevalence higher than 1% was more common at altitudes below 350 m (x2 = 5.21, df = 1, p < 0.05) and at average annual temperatures higher than 9.5 ◦ C (x2 = 4.3, df = 1, p < 0.05). The distribution model (Fig. 2) has shown a medium prediction (AUC = 0.614) (Fig. S1 in Supplementary file). The altitude had the highest influence on the distribution (Fig. S2 in Supplementary file), the presence of A. phagocytophilum being negatively associated with the altitude (Supplementary file S2). Discussion Only few scattered data concerning the epidemiology of A. phagocytophilum in Romania are available. The present study is the first survey on the presence of A. phagocytophilum in questing ticks from this country. In I. ricinus ticks collected from
Please cite this article in press as: Matei, I.A., et al., Anaplasma phagocytophilum in questing Ixodes ricinus ticks from Romania. Ticks Tick-borne Dis. (2015), http://dx.doi.org/10.1016/j.ttbdis.2015.03.010
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domestic hosts in three counties from the southern part and one from the northern part of the country, the prevalence of A. phagocytophilum was 6.7% (Ionit¸a˘ et al., 2013). A study on the prevalence of A. phagocytophilum in I. ricinus collected from roe deer and goats in two counties from southern Romania found a prevalence of 1.4% (P˘aduraru et al., 2012). Another study was performed on I. ricinus collected from hedgehogs (Erinaceus roumanicus) by Dumitrache et al. (2013), where the authors found 12% prevalence. Neither of these studies evaluated the seasonal variation. Moreover, no evaluation on the host infection was performed. Interestingly, Pas¸tiu et al. (2012) found a relatively high prevalence (18.8%) of A. phagocytophilum in Hyalomma aegyptium ticks collected from tortoises (Testudo graeca) in southeastern Romania, suggesting a rather passive carrier role of these ticks but also an epidemiological proof for their host switching behavior (Pas¸tiu et al., 2012). Additionally, a recent study from western Romania has provided evidence for the role of wild boars (Sus scrofa) as potentially important reservoir hosts for A. phagocytophilum (Kiss et al., 2014). No data on human clinical cases have been published so far in Romania. A. phagocytophilum has been detected in ticks from all over the world, mostly in the northern hemisphere (Strle, 2004; Teglas and Foley, 2006). In Europe it was recorded from almost all countries with prevalence ranging between 0.5% and 34% (Christova et al., 2001; Egyed et al., 2012). The overall prevalence we recorded falls within the European average (Strle, 2004). However, the nationwide A. phagocytophilum prevalence may be lower since the locations taken in our study had considerable forest coverage and previous studies have shown a higher prevalence in this type of habitat (Halos et al., 2006, 2010). In each country and between countries the prevalence seems to be variable. In Central and Eastern Europe, high prevalence variability was found in the different countries, from low values in Hungary and Republic of Moldova (0.5–2.4%), medium values in Slovakia and Russia (8–9%) to high values of 34% in Bulgaria (Christova et al., 2001; Masuzawa et al., 2008; Egyed et al., 2012; Subramanian et al., 2012; Movila et al., 2013). Several factors were suggested to explain these local variations in prevalence, the most commonly incriminated being the abundance and population structure of the tick vector and the abundance and diversity of potential reservoir hosts. The population ecology of the vector ticks depends on complex factors, such as the type of habitat, climatic conditions (related mostly to latitude and altitude) but also the host availability (Gern and Humair, 2002). Population structure and abundance of the vector depend also on the seasonal activity, I. ricinus being more active in the spring (Gern and Humair, 2002); hence, the ratio of adults and nymphs will influence the overall prevalence. More studies have shown differences in the prevalence of A. phagocytophilum between developmental stages, which is higher in adults than in nymphs. Our results have also shown a higher prevalence of A. phagocytophilum in adults compared to nymphs (5.56% vs. 0.69%). Similar results were recorded in other European countries (Christova et al., 2001; Overzier et al., 2013; Venclikova et al., 2014; Granquist et al., 2014). These differences in A. phagocytophilum prevalence could be explained by the higher probability of the adults to feed on an infected reservoir host, especially since transovarial transmission of A. phagocytophilum has not yet been demonstrated. Among adult ticks, females seem to be more often infected with A. phagocytophilum (Rosef et al., 2009). Although the sampling in the current study was done exclusively during the spring, previous studies have shown little or no significant seasonal variation in the prevalence of A. phagocytophilum (Silaghi et al., 2008). However, others found contradicting results: higher prevalence in spring (Mysterud et al., 2013) or higher prevalence during autumn (Reye et al., 2010). Geographical patterns and habitat structure were demonstrated as important factors not only for the abundance of ticks, but also for
the prevalence of A. phagocytophilum infection in vectors. Various studies reported a focal distribution of the infection, with significant differences between urban parks, pasture and forest (Ogden et al., 1998; Wielinga et al., 2006; Halos et al., 2006, 2010). The prevalence in ticks may also be influenced by the A. phagocytophilum epidemiology in the reservoir hosts, given that different developmental stages of ticks may have different preferred host species (Feider, 1965; Mihalca et al., 2012b). In an extensive study regarding the distribution of ticks and host associations in Romania, some patterns were observed for Ixodes ricinus (Mihalca et al., 2012a,b). For instance, on the majority of rodent species, exclusively immature stages of I. ricinus were observed. Adult I. ricinus were found only on three rodent species (Apodemus agrarius, Microtus arvalis and Micromys minutus). On insectivores, wild ruminants and humans, all stages were observed, while on wild carnivores, only nymphs and adults were found. Wild boars (Sus scrofa) were infested only with adults (Mihalca et al., 2012b). Similar observations were made for birds, adults being found only in few instances (Mihalca et al., 2012b; Sándor et al., 2014). An interesting aspect was the correlation with the average altitude, with a higher presence of A. phagocytophilum in localities below 350 m altitude (x2 = 5.21, df = 1, p < 0.03). This result is similar with previous observations, according to which A. phagocytophilum prevalence is negatively correlated with altitude. In the study of Grøva et al. (2011) a smaller prevalence of A. phagocytophilum infection was observed in lambs grazing at high altitude compared with farms grazing at low altitude. Additionally Gilbert (2010) found that the density of I. ricinus is decreasing at increasing altitudes. Furthermore, we found a positive correlation with the local average annual temperature. Higher prevalence was found at an average temperature exceeding 9.5 ◦ C (x2 = 4.3, df = 1, p < 0.04). Influence of the altitude and temperature on the prevalence of A. phagocytophilum could be explained by the lower vector abundance (Gilbert, 2010). Differences in reservoir hosts communities could also influence the prevalence of A. phagocytophilum in questing ticks from the same habitats. Gilbert (2010) tested the effect of different hosts on tick abundance depending on the altitude. Her models have shown an increasing abundance of larvae and nymphs with the increasing deer abundance. The effect of deer abundance on nymphs was significant at lower altitudes and not significant at high altitudes. Moreover, a negative correlation was observed between the deer abundance and altitude. For the mountain hare abundance, a negative trend on larval abundance was observed, as the relation between mountain hare and altitude was positive (Gilbert, 2010). Similarly, the structure of rodent and bird communities, which are potential reservoir and dispersal hosts for A. phagocytophilum (Rejmanek et al., 2011; Capligina et al., 2014; Lommano et al., 2014) may also differ at various altitudes and temperatures (Wagner, 1974, 1976). In a previous study, the ankA gene analysis has shown a correlation between host species and the four identified A. phagocytophilum ankA gene clusters (Scharf et al., 2011). Sequences from dogs, humans, horses and cats were found exclusively in cluster I. Overall, sequences from ruminants were diverse and were found in all clusters, but particularly, sequences from roe deer were found exclusively in clusters II and III, while other ruminants (e.g. red deer, sheep, bison, cows) were mainly in clusters I and IV (Scharf et al., 2011). In the present study, phylogenetic tree analysis has shown two genetic lineages. One contains the sequences RO1 (KP164415), RO2 (KP164412) and RO4 (KP164414) and sequences isolated from ticks and animal tissues which were previously classified in cluster I. The second one includes the sequence RO3 (KP164413) and sequences isolated from ruminant tissues, mainly roe deer. These results suggest a large variety of host species for A. phagocytophilum in Romania. Wild boars as host for A. phagocytophilum were previously studied (Kiss et al., 2014), but further analysis regarding wild
Please cite this article in press as: Matei, I.A., et al., Anaplasma phagocytophilum in questing Ixodes ricinus ticks from Romania. Ticks Tick-borne Dis. (2015), http://dx.doi.org/10.1016/j.ttbdis.2015.03.010
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ruminants, domestic carnivores and horses are required in order to understand the epidemiology of A. phagocytophilum in Romania. According to the distribution model, most of Romania’s territory has typical conditions for the presence of A. phagocytophilum, but the risk is generally moderate. The risk of acquiring A. phagocytophilum associated with I. ricinus bite is lower in mountainous areas, as it is negatively correlated with altitude. Acknowledgments ADM, ADS, CD and GD were supported in this research by a grant from the UEFISCDI grant PCE 236/2011. CM was supported by a grant from USAMV 1215/28/2012. CD, ZK, DIM and MOD’s work was financed by POSDRU grant no. 159/1.5/S/136893 grant with title: “Parteneriat strategic pentru cres¸terea calit˘at¸ii cercet˘arii s¸tiint¸ifice din universit˘at¸ile medicale prin acordarea de burse doctorale s¸i postdoctorale – DocMed.Net 2.0”. ADM, MOD, ZK, IAM, AMI, GD and ADS are members of the COST Action TD1303 “European Network for Neglected Vectors and Vector-Borne Infections (EURNEGVEC)”. We would like to thank our colleagues Mircean Viorica, Adriana Györke, Ioana Pas¸tiu and Miruna Oltean for their help during the field work. Special thanks to Friederike von Loewenich for all her valuable advices during the preparation of this manuscript. This paper was published under the frame of EurNegVec COST Action TD1303. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.ttbdis.2015.03.010. References Aktas, M., Altay, K., Dumanli, N., 2011. Molecular detection and identification of Anaplasma and Ehrlichia species in cattle from Turkey. Ticks Tick Borne Dis. 2, 62–65. André, M.R., Baccarim Denardi, N.C., Marques de Sousa, K.C., Gonc¸alves, L.R., Henrique, P.C., Grosse Rossi Ontivero, C.R., Lima Gonzalez, I.H., Vaz Cabral Nery, C., Romeiro Fernandes Chagas, C., Monticelli, C., Alexandre de Santis, A.C.G., Machado, R.Z., 2014. Arthropod-borne pathogens circulating in freeroaming domestic cats in a zoo environment in Brazil. Ticks Tick Borne Dis., http://dx.doi.org/10.1016/j.ttbdis.2014.03.011. André, M.R., Dumler, J.S., Scorpio, D.G., Teixeira, R.H.F., Allegretti, S.M., Machado, R.Z., 2012. Molecular detection of tick-borne bacterial agents in Brazilian and exotic captive carnivores. Ticks Tick Borne Dis. 3, 247–253. Briciu, V.T., Titilincu, A., T˘at¸ulescu, D.F., Carstina, D., Lefkaditis, M., Mihalca, A.D., 2011. First survey on hard ticks Ixodidae collected from humans in Romania: possible risks for tick-borne diseases. Exp. Appl. Acarol. 54, 199–204. Cao, W.C., Zhoa, Q.M., Zhang, P.H., Dumler, J.S., Zhang, X.T., Fang, L.Q., Yang, H., 2000. Granulocytic Ehrlichiae in Ixodes persulcatus ticks from an area in China where lyme disease is endemic. J. Clin. Microbiol. 38, 4208–4210. Capligina, V., Salmane, I., Keiˇss, O., Vilks, K., Japina, K., Baumanis, V., Ranka, R., 2014. Prevalence of tick-borne pathogens in ticks collected from migratory birds in Latvia. Ticks Tick Borne Dis. 5, 75–81. Caturegli, P., Asanovich, K.M., Walls, J.J., Bakken, J.S., Madigan, J.E., Popov, V.L., Dumler, J.S., 2000. AnkA: an Ehrlichia phagocytophila group gene encoding a cytoplasmic protein antigen with ankyrin repeats. Infect. Immun. 68, 5277–5283. Chen, S.M., Dumler, J.S., Bakken, J.S., Walker, D.H., 1994. Identification of a granulocytotropic Ehrlichia species as the etiologic agent of human disease. J. Clin. Microbiol. 32, 589–595. Christova, I., Schouls, L., van de Pol, I., Park, J., Panayotov, S., Lefterova, V., Kantardjiev, T., Dumler, J.S., 2001. High prevalence of granulocytic Ehrlichiae and Borrelia burgdorferi sensu lato in Ixodes ricinus ticks from Bulgaria. J. Clin. Microbiol. 39, 4172–4174. Donit¸a˘ , N., Popescu, A., Pauc˘a-Com˘anescu, M., Mih˘ailescu, S., Biris¸, I.A., 2005. Habitats from Romania. Tehnica Silvica, Bucharest, pp. 377–384 (in Romanian). Dumitrache, M.O., Pas¸tiu, A.I., Kalmár, Z., Mircean, V., Sándor, A.D., Gherman, C.M., Cozma, V., 2013. Northern white-breasted hedgehogs Erinaceus roumanicus as hosts for ticks infected with Borrelia burgdorferi sensu lato and Anaplasma phagocytophilum in Romania. Ticks Tick Borne Dis. 4, 214–217. Dumler, J.S., Barbet, A.F., Bekker, C.P.J., Dasch, G.A., Palmer, G.H., Ray, S.C., Rikihisa, Y., Rurangirwa, F.R., 2001. Reorganization of genera in the families Rickettsiaceae and Anaplasmataceae in the order Rickettsiales: unification of some species of Ehrlichia with Anaplasma. Cowdria with Ehrlichia, and Ehrlichia with Neorickettsia, descriptions of six new species combinations and designation of Ehrlichia
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