Exp Appl Acarol DOI 10.1007/s10493-015-9941-0

Seasonal analysis of Rickettsia species in ticks in an agricultural site of Slovakia Eva Sˇpitalska´1 • Michal Stanko2,3 • Ladislav Mosˇansky´3 Jasna Kraljik3,4 • Dana Miklisova´3 • Lenka Mahrı´kova´2 Martin Bona5 • Ma´ria Kazimı´rova´2

• •

Received: 27 February 2015 / Accepted: 12 June 2015 Ó Springer International Publishing Switzerland 2015

Abstract Many rickettsiae of the spotted fever group are emerging pathogens causing serious diseases associated with vertebrate hosts. Ixodidae ticks are known as their vectors. Investigation of the relative abundance of questing Ixodes ricinus and their infection with Rickettsia spp. in an agricultural site comprising a game reserve in Slovakia was the aim of this study. In total, 2198 I. ricinus (492 larvae, 1503 nymphs and 203 adults) were collected by flagging the vegetation along 100 m2 transects in Rozhanovce (eastern Slovakia): 334, 595 and 1269 in 2011, 2012 and 2013, respectively. Considering questing nymphs and adults, the highest relative density of 81 individuals/100 m2 was observed in May 2013, the lowest of 0.3 individuals/100 m2 in March 2012. A total of 1056 ticks (853 nymphs, 100 females and 103 males; 2011: n = 329, 2012: n = 509 and 2013: n = 218) were individually screened by PCR-based methods for the presence of Rickettsia spp. The overall prevalences were 7.3 % for nymphs, 15 % for females, 7.8 % for males; 7.0 % in 2011, 8.4 % in 2012, and 8.7 % in 2013. The maximum prevalences were observed in July in nymphs and in May in adults. Sequencing showed infection with R. helvetica in 73 ticks (72.6 % nymphs, 16.4 % females, 11 % males) and with R. monacensis in 11 ticks (8 nymphs, 3 females). The results showed the circulation of pathogenic Rickettsia species in the agricultural site and a potential risk for humans to encounter infected ticks. Keywords Ixodes ricinus  Rickettsia helvetica  Rickettsia monacensis  Agricultural site  Rozhanovce & Eva Sˇpitalska´ [email protected] 1

Institute of Virology, Slovak Academy of Sciences, Du´bravska cesta 9, 845 05 Bratislava, Slovakia

2

Institute of Zoology, Slovak Academy of Sciences, Du´bravska cesta 9, 845 06 Bratislava, Slovakia

3

Institute of Parasitology, Slovak Academy of Sciences, Hlinkova 3, 040 01 Kosˇice, Slovakia

4

Department of Zoology, Faculty of Natural Sciences, Comenius University, Mlynska´ dolina B-1, 842 15 Bratislava, Slovakia

5

Department of Anatomy, Faculty of Medicine, Pavol Jozef Sˇafa´rik University, 040 01 Kosˇice, Slovakia

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Introduction Ticks are among the most important vectors of human and animal diseases. They can transmit fungi, viruses, bacteria and protozoa. Five genera and 22 species of soft and hard ticks (Acari: Ixodidae) were recognized in the fauna of Slovakia, from which six species, Ixodes ricinus, Dermacentor reticulatus, D. marginatus, Haemaphysalis concinna, H. inermis and H. punctata are exophilic (Slova´k 2010; Bona and Stanko 2013). Ixodes ricinus is the most widespread tick species in Europe and also in Slovakia. The species is primarily associated with shrubs and deciduous and mixed forests, with a high abundance of small, medium, and large wild vertebrate hosts. During the last few decades, its occurrence has expanded in altitude and latitude and to city parks, gardens, and cemeteries (Medlock et al. 2013; Rizzoli et al. 2014). Ixodes ricinus is an important vector for many pathogens, such as the tick-borne encephalitis virus, Borrelia burgdorferi sensu lato, Anaplasma phagocytophilum, Candidatus Neoehrlichia mikurensis, Rickettsia species, Babesia divergens, Babesia venatorum and Babesia microti (Rizzoli et al. 2014). Rickettsiae are obligate, aerobic, gram-negative, intracellular bacterial parasites of eukaryotes with a worldwide expansion. In Europe, Rickettsia felis, R. typhi, R. prowazekii, R. akari, R. conorii, R. slovaca, R. sibirica mongolotimonae, R. raoultii, R. massiliae, R. aeschlimanni, R. helvetica, and R. monacensis have been implicated in human diseases or reported as emerging pathogens or isolated from vectors or humans (Radulovic et al. 1996; Blanco and Oteo 2006; Oteo and Portillo 2012; Parola et al. 2013). Ixodes ricinus ticks are known to carry mainly R. helvetica and R. monacensis with prevalences ranging from 0.5 to 82.9 %, depending on, e.g., locality, questing or feeding ticks, and developmental stages (Oteo and Portillo 2012; Rizzoli et al. 2014). Rickettsia helvetica was isolated for the first time in 1979 from I. ricinus, but its description as a different species of the spotted fever group was confirmed only in 1993 (Beati et al. 1993). The prevalence rate of R. helvetica infection in ticks was found to vary from 0.5 % in a bird conservation island named Greifswalder Oie in the Baltic Sea to 66 % in The Netherlands (Franke et al. 2010; Sprong et al. 2009). Infections were recorded in questing ticks throughout Europe (Oteo and Portillo 2012). Ixodes ricinus is a major reservoir for R. helvetica. Rodents, other small mammals and roe deer may act as reservoir hosts for R. helvetica, but red deer are not suitable hosts for rickettsia (Sˇpitalska´ et al. 2008; Sˇtefanidesova´ et al. 2008; Sprong et al. 2009; Schex et al. 2011). Human cases caused by R. helvetica with various clinical symptoms have been reported from Sweden, France, Switzerland, and Italy (Nilsson et al. 1999, 2010; Fournier et al. 2000, 2004; Baumann et al. 2003; Nilsson 2009). Rickettsia monacensis was originally isolated as a new species from I. ricinus collected in a city park in Germany (Simser et al. 2002). Phylogenetic analyses of the 16S rRNA, gltA, and rompA gene sequences demonstrated its close relationship with Candidatus Rickettsia sp. IRS3 and Cand. Rickettsia sp. IRS4 isolated from I. ricinus in Slovakia (Sekeyova´ et al. 2000). It is also widespread throughout Europe (Oteo and Portillo 2012). The prevalence rate of R. monacensis in questing ticks varies from 0.5 % in Germany to 37.6 % in Turkey (Silaghi et al. 2008; Gargili et al. 2012). Human cases of R. monacensis infections were reported from Spain (Jado et al. 2007). However, R. massiliae, R. felis and R. typhi were also detected in I. ricinus ticks (Ferna´ndez-Soto et al. 2006; Dobler and Wo¨lfel 2009). Habitat structure and representation of tick maintenance hosts as well as of reservoir hosts of tick-borne pathogens have been shown to greatly affect tick abundance, infection rates in ticks and the risk for humans and domestic animals to be bitten by potentially infected ticks. In Slovakia, R. helvetica and R. monacensis-positive ticks were found in urban areas (parks and cemeteries), a mountain forest and recreational areas mainly in southwestern and northern Slovakia (Sekeyova´ et al. 2000, 2012; Sˇpitalska´ et al. 2014;

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Sˇvehlova´ et al. 2014). However, information about the occurrence of ticks and tick-borne pathogens in rural habitats of Slovakia is limited. Thus, in this study we aimed to observe year-to-year and seasonal variation in relative abundance of questing ticks in the agricultural site Rozhanovce (eastern Slovakia) and the seasonal occurrence of Rickettsia species in questing ticks.

Materials and methods Study site Rozhanovce is an agricultural site in eastern Slovakia with fragmented habitats comprising patches of oak-hornbeam forest and a few cultivated fields and meadows. The study site is located in the vicinity of Rozhanovce village (Kosˇicka´ kotlina basin; 215 m a.s.l.; 48°450 0000 N; 21°210 0000 E), within a special facility for breeding of mouflon (Ovis musimon) and fallow deer (Dama dama) in a game reserve of an extent of 500 ha.

Collection of ticks Three 100 m2 transects were selected within the site. The sampling lines were laid parallel to edges of oak–hornbeam woods and fields. Ticks were flagged with a 1 m2 flannel blanket along transects in monthly intervals, preferably during the season of the highest activity (April–June) and in September–October of 2011–2013. Ticks were stored in 70 % ethanol and identified to species and developmental stage according to Filippova (1977).

DNA extraction and molecular identification Part of the ticks (at least 30 nymphs and 30 adults per collection date, whenever sufficient numbers of ticks were available) were selected for DNA extraction. Genomic DNA was extracted from individual ticks by the NucleoSpinÒ Tissue Macherey–Nagel kit according to manufacturer’s instructions. Detection of Rickettsia spp. was performed using the genusspecific primers RpCS.877p–RpCS.1258n amplifying a 381-bp part of the gltA gene (Regnery et al. 1991). PCR amplifications were carried out using the DyNAzymeTM PCR Master Mix (Finnzymes, Finland) as recommended by the manufacturer on a PTC-200 Peltier Thermal Cycler. The DNA from uninfected ticks and sterile water, and DNA from R. helvetica originating from ticks were used as negative and positive controls, respectively. PCR products were analyzed by electrophoresis in a 1 % agarose gel, stained with GelRedTM (Biotium, Hayward, CA, USA) and visualized with a UV transilluminator (3UV Benchtop Transilluminator LMS-20; UVP, Cambridge, UK). All amplicons were purified and sequenced by Macrogen (The Netherlands; http://www.macrogen.com). DNA sequences were compared with available databases in GenBank using the Basic Local Alignment Search Tool (BLAST) on http://blast.ncbi.nlm.nih.gov/.

Statistical analysis Statistical analysis to test the differences between the prevalences in nymphs and adults was carried out with the v2 test (a = 0.05) using Past version 2.17b software (Hammer et al. 2001). 95 % Confidence intervals (CI) were calculated individually for each

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proportion. Fisher exact test was used to compare prevalence of Rickettsia spp. between seasons, because the condition of v2 goodness-of-fit test was not fulfilled. Correlation between relative abundance of ticks and prevalence of Rickettsia spp. was tested by nonparametric Spearman correlation coefficient. The software STATISTICA 12 (http:// www.statsoft.com) was used for this analysis.

Results Tick relative abundance In total, 2198 I. ricinus (492 larvae, 1503 nymphs and 203 adults) were collected by flagging the vegetation along 100 m2 transects: 334, 595 and 1269 in 2011, 2012 and 2013, respectively. Considering questing nymphs and adults, the highest relative abundance of 81 individuals/100 m2 was observed in May 2013, the lowest 0.3 individuals/100 m2 in March 2012. Ixodes ricinus nymphs and adults displayed a bimodal activity pattern (Fig. 1). Peaks of adults could be observed from April to May and from September to October with a strong dominance of spring activity. The major peaks of nymphs could be observed from May to July and from September to October, also with a strong dominance of spring activity. In 2011, only a few larvae were collected in September, whereas larvae were present in May– July of 2012 and 2013 and also in September of 2013.

Molecular identification of Rickettsia spp.

No. of collected cks/100 m 2

A total of 1056 ticks (853 nymphs, 100 females and 103 males; 2011: n = 329, 2012: n = 509 and 2013: n = 218) were individually screened by PCR-based methods for the presence of Rickettsia spp. The total Rickettsia spp. infection rate of ticks was found to be 8.0 % (CI 6.3–9.6 %) (Table 1). The total infection rate of adult ticks was 11.3 % (CI 6.9–12.7 %) with 7.8 % (CI 2.5–13.0 %) infected males and 15 % (CI 7.9–22.1 %) infected females. Of the nymphs 7.2 % (CI 5.4–8.9 %) were infected with Rickettsia spp. The prevalence in adults 100 90 80 70 60 50 40 30 20 10 0

Larvae

Nymphs

Adults

Month_year Fig. 1 Relative abundance of Ixodes ricinus adults, nymphs, and larvae per 100 m2 in Rozhanovce from April 2011 to September 2013

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0 (0/0)

0 (0/1)

0 (0/1)

0 (0/1)

Males

Females

Total adults

Total

a

6.5 (9/138)

0 (0/0)a

Nymphs

9.2 (27/295)

7.0 (10/70)

16.7 (6/36)

11.8 (4/34)

7.6 (17/225)

May

% positive ticks (no. Rickettsia-positive ticks/no. tested ticks)

7.9 (18/228)

10.0 (9/90)

15.0 (6/40)

6.0 (3/50)

April

March

6.8 (16/237)

9.5 (2/21)

13.3 (2/15)

0 (0/6)

6.5 (14/216)

June

11.2 (11/98)

0 (0/9)

0 (0/4)

0 (0/5)

12.4 (11/89)

July

Table 1 Seasonal occurrence of Rickettsia-infected questing Ixodes ricinus in Rozhanovce in 2011–2013

7.7 (2/26)

0 (0/0)

0 (0/0)

0 (0/0)

7.7 (2/26)

August

4.3 (4/94)

25.0 (1/4)

100 (1/1)

0 (0/3)

3.3 (3/90)

September

7.8 (6/77)

12.5 (1/8)

0 (0/3)

20.0 (1/5)

7.2 (5/69)

October

8.0 (84/1056)

11.3 (23/203)

15.0 (15/100)

7.8 (8/103)

7.2 (61/853)

Total

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was significantly higher than in nymphs (p = 0.048). There was no significant difference in infection rates between female and male ticks (p = 0.10). The overall prevalences of Rickettsia spp. in questing I. ricinus ticks were 6.7 % (22/ 329; CI 4.0–9.4 %; 6.8 % nymphs, 6.1 % females, 6.7 % males) in 2011, 8.5 % (43/509; CI 6.0–10.9 %; 7.4 % nymphs, 18.2 % females, 11.6 % males) in 2012, and 8.7 % (19/ 218; CI 5.0–12.5 %; 7.1 % nymphs, 20.6 % females, 3.3 % males) in 2013. There was no statistically significant difference in total infection rates among years. Table 1 lists the detailed results of seasonal tick infections with Rickettsia spp. during 2011–2013. Analysis of seasonal distribution of Rickettsia-infected I. ricinus showed high occurrence of infected ticks during spring months April–June (6.8–9.2 %) and only one major peak in July. In July, the highest infection rate of 11.2 % was observed, followed by a decreasing infection rate to a minimum of 4.3 % in September. A growing infection rate was followed by a lower autumn peak in October with 7.8 % Rickettsia-infected ticks, with prevalences similar to those recorded in the spring. But there was no significant difference between infection rates in July and September (p = 0.072) nor between September and October (p = 0.33). In nymphs, the maximum prevalence was in July, but in adults it was in May (September and October were not considered in analyses because of low number of collected and tested adults). The variability in adult and nymphal tick infection rates with Rickettsia spp. during the investigation period is shown in Fig. 2. The highest prevalences of Rickettsia-positive questing adults were recorded in May, September and June in 2011, 2012 and 2013, respectively. The highest prevalences of Rickettsia-positive questing nymphs were recorded in July, October, August in 2011, 2012 and 2013, respectively. There was a significant correlation between relative abundance and prevalence of Rickettsia spp. infection in I. ricinus (adults and nymphs) (Spearman R = 0.492, N = 19, p = 0.05). However, the significant correlation was not confirmed for adults and nymphs, when analyzed separately. We also evaluated the seasonal variability in Rickettsia spp. infection rates in questing I. ricinus ticks by comparing two periods: April–July and August–October. The percentage

100 90 80 70 60 50 40 30 20 10 0

Month_year Fig. 2 Relative abundance of Ixodes ricinus (numbers of adults and nymphs per 100 m2) per month (bars) and prevalence (%) of Rickettsia-positive ticks (dots), in Rozhanovce from April 2011 to September 2013

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of infected ticks did not differ significantly between these two periods (Fisher exact tests, p [ 0.05) (Table 2). In 73 (86.9 %) of the 84 Rickettsia spp. positive ticks, DNA sequencing showed infection with R. helvetica (72.6 % nymphs, 16.4 % females, 11.0 % males) and in 11 (13.1 %) with R. monacensis (8 nymphs, 3 females). Both identified rickettsiae, R. helvetica and R. monacensis, occurred in ticks during the whole season.

Discussion The hard tick I. ricinus is associated with several symbiotic and pathogenic microorganisms, and plays an important role as a vector for these pathogens. We aimed to study ticks and tick-borne pathogenic Rickettsia species in an agricultural site in eastern Slovakia. We collected 2198 I. ricinus ticks during a 3-year study and identified the presence of two pathogenic species, R. helvetica and R. monacensis. These species are facultative pathogens of humans and animals (Nilsson et al. 1999, 2010; Fournier et al. 2004; Jado et al. 2007). The present study is the first monitoring of the presence of rickettsial bacteria in the Rozhanovce site. Previous studies investigated rickettsial infections in ticks in urban, suburban or natural areas in Slovakia. R. helvetica and R. monacensis were identified in I. ricinus ticks in south-western and central parts of the country, i.e., urban parks in Bratislava, Malacky and Martin, natural forest in the Martinske´ Hole Mts. and a floodplain forest at Vojka nad Dunajom. In the previously investigated sites DNA of Rickettsia spp. was identified in 9–11.7 % of tested ticks (Sˇpitalska´ et al. 2014; Sˇvehlova´ et al. 2014). In the present study the overall prevalence of Rickettsia spp. was 8 %. No significant differences between prevalences of infection in tick females versus males were found in the previous studies, which is in agreement with results of this study. However, the prevalence in adults was significantly higher than in nymphs (Sˇpitalska´ et al. 2014), comparable with

Table 2 Differences in prevalence of Rickettsia spp. in questing Ixodes ricinus between two seasonal peaks of activity in Rozhanovce (April–July and August–October) April–July Prevalence (%) (no. posit/total)

August–October 95 % CI

Prevalence (%) (no. posit/total)

p (Fisher exact test) 95 % CI

2011 Nymphs

7.4 (17/231)

4.3–11.5

2.9 (1/35)

0.1–14.9

0.48

Adults

6.7 (4/60)

1.8–13.6

0 (0/3)

0.0–70.8

1.0

Total

7.2 (21/291)

4.5–10.8

2.6 (1/38)

0.1–13.8

0.49

2012 Nymphs Adults Total

7.6 (24/317) 13.2 (9/68)

4.9–11.1 6.2–23.7

6.9 (8/116) 25.0 (2/8)

3.0–13.1

1.0

3.2–65.1

0.33

3.9–14.3

1.0

8.6 (33/385)

6.0–11.8

8.1 (10/124)

Nymphs

10.0 (12/120)

5.3–16.8

2.9 (1/34)

0.1–15.3

0.30

Adults

12.7 (8/63)

5.6–23.5

0 (0/1)

0.0–97.5

1.0

Total

10.9 (20/183)

6.8–16.4

2.9 (1/35)

0.1–14.9

0.21

2013

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this study. Still, in the current study, the differences could also be due to the portion of tested ticks, as all adult ticks, but only part of the nymphs were analysed. It is difficult to compare the prevalence of rickettsiae in I. ricinus ticks in an agricultural site in Slovakia with other agricultural sites in Europe. To the best of our knowledge, there is only one study on an agricultural site in Europe, i.e., in Germany (Overzier et al. 2013). The majority of investigations on prevalence of tick-borne pathogens in Central Europe focused on urban, suburban and natural habitats. Overzier et al. (2013) collected I. ricinus in a pasture in Bavaria and found 13.3 % adults and 15.7 % nymphs Rickettsia spp. positive. R. helvetica and R. monacensis were identified, but significantly higher prevalence for Rickettsia spp. was found in ticks collected in a natural area in Bavaria than on the pasture. During 2011–2013, the annual prevalence of rickettsiae in Rozhanovce ranged from 6.7 to 8.7 %. Sprong et al. (2009) also reported that the annual prevalence of R. helvetica in all three tick developmental stages was practically constant in The Netherlands for 9 years (2000–2008). In contrast, Hildebrandt et al. (2010) recorded year-to-year variation in prevalences in Germany, with infection rate in 2006 (19.2 %) almost two-fold higher than in 2007 (10.1 %). Seasonal variation of Rickettsia-infected questing I. ricinus ticks has been widely studied in Germany (Silaghi et al. 2008; Hildebrandt et al. 2010; Schorn et al. 2011; Tappe and Strube 2013), Denmark (Kantso et al. 2010), The Netherlands (Sprong et al. 2009), and now in Rozhanovce. In our study, seasonal variation of Rickettsia-infected I. ricinus differed among years. However, in general high prevalence of infected ticks was recorded during spring months April–June (6.8–9.2 %) with a peak in July (infection rate of 11.2 %), followed by a decreasing infection rate to a minimum of 4.3 % in September. An increasing infection rate was followed by a lower autumn peak in October with 7.8 % Rickettsia-infected ticks. But the differences between infection rates in July and September and between September and October were not significant. The maximum prevalence in nymphs was recorded in July, in adults it was in May (September and October were not considered because of the low number of collected and tested adults). In Germany, 1-year studies of seasonal variation in prevalence of Rickettsia-infected I. ricinus were performed. Silaghi et al. (2008) presented that the monthly variation was not significant in ticks collected in southern Germany during 2006. But, according to Hildebrandt et al. (2010), the number of ticks (both nymphs and adults) infected with Rickettsia spp. increased in summer (July and August) in Central Germany. Schorn et al. (2011) observed in Bavarian public parks in Germany in 2009 that the minimum prevalences in April (4.0 %) and September (3.5–6.6 %) were significantly lower than in June (7.7–8.6 %) and July (8.7–10.2 %). An analysis of seasonal Rickettsia spp. distribution in the city of Hanover in 2010 showed two major peaks, in June and October. In June, the highest infection rate was 31.7 %, followed by a decreasing infection rate to a minimum of 19.7 % in August. A growing infection rate in September was followed by a second peak in October with 31.0 % Rickettsia-infected ticks. Statistically significant differences between infection rates in June vs. August as well as August vs. October were determined (Tappe and Strube 2013). In contrast, Kantso et al. (2010) recorded that the rickettsial load in tick samples from Denmark did not significantly differ among collection dates. Similarly, in The Netherlands the monthly infection rate of nymphs did not change significantly (Sprong et al. 2009). The different data about prevalence rates, seasonal variation of Rickettsia-infected ticks and representation of Rickettsia species may have been influenced by many factors, e.g. tick phenology, sex ratio, habitat type, microclimate, season of tick collection, presence

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and diversity of tick host species. Still, this is the first report on the identification of two human pathogens, R. helvetica and R. monacensis, in ticks from an agricultural site in eastern Slovakia. The data about the presence of the two rickettsial pathogens in ticks indicate their potential epidemiological and epizootological significance and are essential for risk assessment and adequate prevention of tick-transmitted diseases in the study area. Acknowledgments The study was partly funded by EU grant FP7-261504 EDENext and is catalogued by the EDENext Steering Committee as EDENext362 (http://www.edenext.eu). The contents of this publication are the sole responsibility of the authors and do not necessarily reflect the views of the European Commission. Financial support of grant APVV DO7RP-0014-11 and Project No. 2/0061/13 from the Scientific Grant Agency of Ministry of Education and Slovak Academy of Sciences are acknowledged. The authors thank Dr. Lucia Blanˇarova´ for her help with flagging of ticks from the vegetation. Conflict of interest No competing financial interest exist. The authors declare no conflict of interest. Ethical standard The experiments presented in this paper comply with current laws of the Slovak Republic. This article does not contain any studies with human participants or animals performed by any of the authors.

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