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Occurrence of Francisella spp. in Dermacentor reticulatus and Ixodes ricinus ticks collected in eastern Poland
Ticks and Tick-borne Diseases 6 (2015) 253–257
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Short communication
Occurrence of Francisella spp. in Dermacentor reticulatus and Ixodes ricinus ticks collected in eastern Poland ˛ a , Anna Sawczyn a , Ewa Cisak a , Jacek Sroka a,b , Angelina Wójcik-Fatla a,∗ , Violetta Zajac a Jacek Dutkiewicz a b
Department of Zoonoses, Institute of Rural Health, Lublin, Poland Department of Parasitology and Invasive Diseases, National Veterinary Research Institute, Pulawy, Poland
a r t i c l e
i n f o
Article history: Received 28 June 2014 Received in revised form 19 December 2014 Accepted 21 January 2015 Available online 31 January 2015 Keywords: Francisella tularensis subsp. holarctica Francisella-like endosymbionts Dermacentor reticulatus Ixodes ricinus Eastern Poland
a b s t r a c t A total of 530 questing Dermacentor reticulatus ticks and 861 questing Ixodes ricinus ticks were collected from Lublin province (eastern Poland) and examined for the presence of Francisella by PCR for 16S rRNA (rrs) and tul4 genes. Only one female D. reticulatus tick out of 530 examined (0.2%) was infected with Francisella tularensis subspecies holarctica, as determined by PCR of the rrs gene. None of 861 I. ricinus ticks were infected with F. tularensis. In contrast, the presence of Francisella-like endosymbionts (FLEs) was detected in more than half of the D. reticulatus ticks (50.4%) and 0.8% of the I. ricinus ticks. The nucleotide sequences of the FLEs detected in D. reticulatus exhibited 100% homology with the nucleotide sequence of the FLE strain FDrH detected in Hungary in D. reticulatus. In conclusion, our results suggest a low contribution of D. reticulatus and I. ricinus ticks to the circulation of F. tularensis in eastern Poland. This finding, however, needs to be confirmed by further studies in other areas. Our study confirmed the common infection of D. reticulatus with Francisella-like endosymbionts (FLEs) of unknown pathogenic potential and revealed, for the first time, a low grade of infection of I. ricinus with FLEs. © 2015 Elsevier GmbH. All rights reserved.
Introduction Francisella tularensis (F. tularensis) is a fastidious, aerobic, gram-negative coccobacillus that affects vertebrates, especially lagomorphs and rodents. In humans, this coccobacillus causes tularemia, a potentially fatal multi-systemic zoonotic disease that occurs in the northern hemisphere, including North America, Europe, and Asia. The species F. tularensis is divided into three subspecies: subsp. Tularensis, which has high virulence and occurs predominantly in North America, subsp. Holarctica, which has mild virulence and occurs throughout the northern hemisphere, and subsp. Mediasiatica, which has mild virulence and occurs in Central Asia (Carvalho et al., 2014). Humans can become infected by F. tularensis by being bitten by hematophagous arthropods (ticks, mosquitos, and deer flies), by direct contact with infected animals (mostly hares and rabbits), by ingestion of contaminated food or water, and by inhalation of infected aerosols. The bacterium is highly infectious and is considered as a potential bioweapon (Carvalho et al., 2014; CDC, 2005;
∗ Corresponding author at: Department of Zoonoses, Institute of Rural Health, Jaczewskiego 2, 20-090 Lublin, Poland. Tel.: +48 817184555; fax: +48 817184555. E-mail address: [email protected] (A. Wójcik-Fatla). http://dx.doi.org/10.1016/j.ttbdis.2015.01.005 1877-959X/© 2015 Elsevier GmbH. All rights reserved.
Foley and Nieto, 2010; Zhang et al., 2008; Franke et al., 2010; Kaysser et al., 2008; Kugeler et al., 2005; Kreizinger et al., 2013). The transovarial transmission of F. tularensis in ticks is controversial and may depend on what subpopulations of F. tularensis and what tick species are present (Petersen et al., 2009). The principal tick vectors include species of the genera Amblyomma, Dermacentor, Haemaphysalis, Ixodes and Ornithodoros (Gordon et al., 1983). Many tick species are also hosts of bacteria that are closely related to F. tularensis, called Francisella-like endosymbionts (FLEs) (Dergousoff and Chilton, 2012). The pathogenic potential of FLEs remains unknown, although sequences homologous to the iglC and mglA genes of F. tularensis, which have been implicated in bacterial pathogenicity, have been detected in FLEs (MachadoFerreira et al., 2009). FLEs appear to replicate intracellularly, and they are transmitted transovarially. To date, there is no evidence of horizontal transmission through tick bites (Ivanov et al., 2011). FLEs are widely distributed, and a number of diverse FLEs have been reported in various tick genera on at least four continents (2011; Scoles, 2004). In Europe, thus far, FLEs have been isolated from Dermacentor reticulatus (D. reticulatus) in Hungary (Kreizinger et al., 2013; Sréter-Lancz et al., 2009), Portugal (De Carvalho et al., 2011), France (Michelet et al., 2013) and Germany (Gehringer et al., 2013) and from Hyalomma marginatum marginatum,
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males, and for nymphs the DNA concentrations ranged from 18 to 80 ng/l. Detection of Francisella spp. DNA in ticks by PCR
Fig. 1. Map of collection of ticks located in the Lublin province, eastern Poland (source of map http://www.polskainfo.pl). Locality A: Ostrów Lubelski; B: Parczew; C: Włodawa; D: Zwierzyniec.
Hyalomma aegyptium, Rhipicephalus sanguineus and D. reticulatus in Bulgaria (Ivanov et al., 2011). No recent reports are available on the occurrence of F. tularensis in potential arthropod vectors in Poland. To fill this gap, we examined samples of D. reticulatus and I. ricinus ticks collected from eastern Poland for the presence of F. tularensis; we also considered the potential for FLEs in the sampled ticks. Materials and methods Collection of ticks A total of 530 questing D. reticulatus ticks (273 females and 257 males) and 861 questing I. ricinus ticks (200 females, 179 males and 482 nymphs) were collected during the spring/summer season in 2011–2012 in 4 localities situated in the Lublin province (eastern Poland). D. reticulatus ticks were collected in 3 localities situated ˛ ´ on the Łeczy nsko-Włodawskie Lakeland: Ostrów Lubelski (locality ‘A’), Parczew (locality ‘B’), and Włodawa (locality ‘C’). I. ricinus ticks were collected in a forest inspectorate, Zwierzyniec (locality ‘D’), situated on the Roztocze Highland (Fig. 1). Ticks were collected by dragging a woolen flag over the lower vegetation and litter along the paths and edges of deciduous and mixed forests. DNA isolation from ticks Total DNA was isolated from the adult ticks separately and from nymphs in pools of 5 specimens (Rijpkema et al., 1996) by boiling in 0.7 M ammonium hydroxide. The concentration of DNA in the isolates was determined with the NanoDrop ND1000 Spectrophotometer (USA). For D. reticulatus, the determined DNA concentrations ranged from 520 to 672 for males and from 670 to 878 ng/l for females. For I. ricinus, the DNA concentrations ranged from 309 to 506 ng/l for females and from 175 to 328 ng/l for
For the 16S rRNA (rrs) and tul4 (encoding 17 kDa lipoprotein) genes, PCR assays were performed according to the methods of Michelet et al. (2013) with some modifications. A 50 l reaction volume was used and contained the following: 1 U Taq DNA polymerase (Qiagen, USA), 1× PCR buffer containing 15 mM MgCl2 , 2 mM dNTP (final concentration 0.2 mM) (Fermentas, Lithuania), 2.5 l 10 M each of primer (Eurogentec, Seraing, Belgium), 2 l of DNA and nuclease-free water (Applied Biosystems, USA). For the rrs and tul4 genes, the primers F11 and F5 (Forsman et al., 1994) and FT393 and FT642 (Long et al., 1993) were applied, respectively. The amplification was carried out in C1000 Thermal Cycler (BioRad, USA). After electrophoresis in a 1.5% agarose gel under standard conditions and staining with ethidium bromide solution (2 g/ml), the products of the 16S rRNA amplification were identified as a 1140 bp band. For the amplification products of tul4, electrophoresis was performed in 2% agarose gels, and a 248 bplong electrophoresis band was considered positive. The F. tularensis strain FSC043 was used as the positive control and was kindly provided by Prof. M. Kondrusik (Department of Infectious Diseases and Neuroinfection in Białystok, Poland). For the negative control, instead of using matrix DNA, we used nuclease-free water. In addition, positive specimens were examined using the lpnA gene, which encodes a region of a 17 kDA lipoprotein that is different from tul4. A 233-bp fragment of lpnA has been recommended as an appropriate marker for differentiating between F. tularensis and FLEs (Forestal et al., 2008; Escudero et al., 2008). The PCR was performed according to the method of Gehringer et al. (2013). After PCR, the samples that contained rrs, tul4 and lpnA genes were classified as positive. DNA sequencing DNA sequencing was performed with an ABI PRISM 310 Genetic Analyzer (Applied Biosystems, Inc., Foster City, CA, USA) using Abi Prism Big Dye Terminator v. 3.1. Cycle Sequencing Kits and Big Dye XTerminator Purification Kits (Applied Biosystems). The same primers as those used for the PCR of the rrs, tul4, lpnA genes were also used for sequencing. The resulting sequences were compared with sequences in the GenBank database using the BLAST server on the National Center for Biotechnology Information website (Bethesda, MD, USA). Statistical analysis The obtained results were analyzed by 2 test and Student’s ttest using the STATISTICA v. 6.0 package (Statsoft, Tulsa, OK, USA). A p value <0.05 was considered statistically significant. Results Only one female D. reticulatus tick out of 530 examined (0.2%) was infected with the F. tularensis subspecies holarctica according to the PCR results for the rrs gene (accession number CP007148). Unfortunately, the sequence analyses of tul4 and lpnA genes were not successful because of the poor quality of the PCR products (the bands on the agarose gel were too weak). In contrast, Francisella-like endosymbionts (FLEs) were detected in more than half of the D. reticulatus ticks examined (50.4%) (Table 1). FLE infection of ticks was significantly dependent on locality (p = 0.0083) and sex: females were infected significantly more often than males (p = 0.0034). None of the 861 I. ricinus ticks
267/530 (50.4%) 91/191 (47.6%) 0/191 (0)
1/530 (0.2%)
154/273 (56.4%) 113/257 (44.0%) 64/96 (66.7%) 27/95 (28.4%) 0/96 (0) 0/95 (0)
1/273 (0.4%) 0/257 (0)
Francisella-like endosymbionts (FLEs) Francisella tularensis ssp. holarctica Francisella-like endosymbionts (FLEs) Francisella tularensis ssp. holarctica
74/191 (38.7%) 102/148 (68.9%) 1/148 (0.7%) Total
0/191 (0)
32/96 (33.3%) 42/95 (44.2%) 58/81 (71.6%) 44/67 (65.7%) 1/81 (1.2%) 0/67 (0) Females Males
0/96 (0) 0/95 (0)
Francisella-like endosymbionts (FLEs) Francisella tularensis ssp. holarctica Francisella tularensis ssp. holarctica
Francisella-like endosymbionts (FLEs)
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Table 2 Prevalence of Francisella tularensis and Francisella-like endosymbionts (FLEs) determined with the genes tul4 and rrs in Ixodes ricinus ticks found on the territory of the Lublin province (eastern Poland). Locality/species
Locality ‘D’ Infected/examined (percent)
Stage
Francisella tularensis ssp. holarctica
Francisella-like endosymbionts (FLEs)
Females Males Nymphs
0/200 (0) 0/179 (0) 0/482 (0)
5/200 (2.5%) 2/179 (1.1%) 0/482 (0)
Total
0/861 (0)
7/861 (0.8%)
were infected with F. tularensis. The presence of Francisella-like endosymbionts (FLEs) was detected in 0.8% of the I. ricinus ticks (Table 2). FLE infection of I. ricinus ticks was significantly dependent on stage. None of the nymphs were infected with FLEs, and this result was significantly lower than that observed in the female (p = 0.0005) and male (p = 0.0214) ticks. Sequence analysis of 30 randomly selected samples of D. reticulatus that were positive for FLEs and seven randomly selected samples of I. ricinus that were positive for FLEs showed 100% homology with the nucleotide sequence of the tul4 gene (GenBank accession no. EU126640) from an FLE detected previously in D. reticulatus in Hungary. The sequence analysis also showed 100% homology of the lpnA gene sequences of the samples with the FLEs with accession numbers FN686808–FN686813, FN686815, FN686816, HM629449, and KJ477082 and 100% homology of the rrs gene sequence of the samples with the FLEs with accession numbers JX561116, JQ942365, HQ705173 and EU234535. Discussion
Stage
Total Infected/examined (percent) Locality ‘C’ Infected/examined (percent) Locality ‘B’ Infected/examined (percent) Locality ‘A’ Infected/examined (percent) Locality/species
Table 1 Prevalence of Francisella tularensis and Francisella-like endosymbionts (FLEs) determined with the genes tul4 and rrs in Dermacentor reticulatus ticks found on the territory of the Lublin province (eastern Poland).
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In this study, for the first time, we report the occurrence of F. tularensis subsp. holarctica and Francisella-like endosymbionts (FLEs) in ticks from Poland. The epidemiological situation of tularemia in Poland is not fully known. The first human case of tularemia was described in 1949, and from 1949 to 2009 a total of 614 cases were reported and were largely attributed to contact with infected hares (Kłapec´ and Cholewa, 2011). In 2012 and 2013, only 6 and 8 cases were reported, respectively (morbidity for 100,000 equal to 0.02) (PZH, 2013). Pancewicz et al. (2004) found a low prevalence of reactions to the F. tularensis antigen serologically among forestry workers (2.06%), casting doubt on the role of the I. ricinus tick in the epidemiology of tularemia in Poland. The results of a study by Rastawicki et al. (2006) showed that antibodies to F. tularensis were detected slightly more often in healthy forest workers than in healthy blood donors, but this difference was not statistically significant. Until now, nothing has been reported on the incidence of tularemia in humans and animals in eastern Poland, which was the location of tick sampling in the present study. The D. reticulatus ticks collected in eastern Poland were infected with the zoonotic bacterium F. tularensis subsp. holarctica at a low rate of 0.2%. This is in accordance with the results of a study in Hungary by Kreizinger et al. (2013), which showed a prevalence of F. tularensis in D. reticulatus ticks equal to 0.27%. Our results are also in accordance with those of Gehringer et al. (2013) from Germany, Bonnet et al. (2013) from France, and Reye et al. (2013) from Belarus, none of whom found F. tularensis subsp. holarctica in D. reticulatus. Our observed rate of infection was lower than that noted by De Carvalho et al. (2007) in Portugal (1.1%). It was also lower than those reported earlier from natural foci of tularemia in the Czech Republic and Austria by Hubálek et al. (1996, 1997, 1998), who found a prevalence of virulent strains of F. tularensis in D. reticulatus equal to 2.3–2.6% and was lower than that reported
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from a natural focus of tularemia in western Slovakia by Gurycová et al. (1995), who found a prevalence ranging from 0.5 to 2%. Hubálek et al. (1997) have the opinion that D. reticulatus plays an important role as a vector of tularemia in endemic areas. In this context, our results suggest that for the examined area of eastern Poland, F. tularensis is not circulating among D. reticulatus ticks to any great extent and its role as a vector of tularemia in this area is unknown. In contrast to F. tularensis, the prevalence of FLEs was high (50.4%). This confirms the reports from other European countries (Kreizinger et al., 2013; Sréter-Lancz et al., 2009; De Carvalho et al., 2011; Michelet et al., 2013; Gehringer et al., 2013) that D. reticulatus is a suitable host for these bacteria. Our result is similar to that reported from Germany (57%) (Gehringer et al., 2013) higher than those reported from Hungary (1.2–3%) (Kreizinger et al., 2013; Sréter-Lancz et al., 2009) and Portugal (39%) (De Carvalho et al., 2011), and lower than that reported from France (79%) (Michelet et al., 2013). It should be mentioned that some of these comparisons might not be accurate because of the differences in the PCR methods used by the individual researchers and the differences in the numbers of ticks tested. None of the I. ricinus ticks examined by us were infected with F. tularensis, and this observation is in agreement with those reported by Sréter-Lancz et al. (2009), Egyed et al. (2012), and Kreizinger et al. (2013) from Hungary, Bonnet et al. (2013) from France, Reye et al. (2010) from Luxembourg and Sanogo et al. (2003) from Italy, who also did not find this bacterium in I. ricinus. Low grade infection rates of I. ricinus with F. tularensis were reported from the Czech Republic (0.2%) (Hubálek et al., 1996), Slovakia (0.3%) (Gurycová et al., 1995), France (0.8%) (Reis et al., 2011), Central Germany (1.2–1.6%) (Franke et al., 2010), Belarus (1.4%) (Reye et al., 2013), and Serbia (3.8%) (Milutinovic´ et al., 2008). A distinctly higher infection rate of I. ricinus with the F. tularensis subsp. holarctica was found by Gehringer et al. (2013) in a territory of endemic focus of tularemia in Baden-Wuerttemberg, Germany. In contrast, not FLEs were detected in 916 I. ricinus ticks. Similarly, in Hungary, of 3222 I. ricinus ticks none were found to be infected with FLEs (Kreizinger et al., 2013). Although the same genetic markers were used for all studies, the results of Kreizinger et al. contrast those of our study, which only showed FLEs in I. ricinus. To the best of our knowledge, this report is the first to describe the presence of Francisella-like endosymbionts (FLEs) in adult I. ricinus ticks. In conclusion, our results suggest a low contribution of D. reticulatus and I. ricinus ticks to the circulation of F. tularensis in eastern Poland. However, this finding needs to be confirmed by further studies in other areas, especially considering the possible occurrence of specific microfoci reported by Goethert and Telford (2009). Our study confirmed the common infection of D. reticulatus with Francisella-like endosymbionts (FLEs) of unknown pathogenic potential and revealed, for the first time the low grade infection of I. ricinus with FLEs.
Acknowledgment This study was supported by the National Science Centre (Grant: N N40404267640).
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Short communication
Occurrence of Francisella spp. in Dermacentor reticulatus and Ixodes ricinus ticks collected in eastern Poland ˛ a , Anna Sawczyn a , Ewa Cisak a , Jacek Sroka a,b , Angelina Wójcik-Fatla a,∗ , Violetta Zajac a Jacek Dutkiewicz a b
Department of Zoonoses, Institute of Rural Health, Lublin, Poland Department of Parasitology and Invasive Diseases, National Veterinary Research Institute, Pulawy, Poland
a r t i c l e
i n f o
Article history: Received 28 June 2014 Received in revised form 19 December 2014 Accepted 21 January 2015 Available online 31 January 2015 Keywords: Francisella tularensis subsp. holarctica Francisella-like endosymbionts Dermacentor reticulatus Ixodes ricinus Eastern Poland
a b s t r a c t A total of 530 questing Dermacentor reticulatus ticks and 861 questing Ixodes ricinus ticks were collected from Lublin province (eastern Poland) and examined for the presence of Francisella by PCR for 16S rRNA (rrs) and tul4 genes. Only one female D. reticulatus tick out of 530 examined (0.2%) was infected with Francisella tularensis subspecies holarctica, as determined by PCR of the rrs gene. None of 861 I. ricinus ticks were infected with F. tularensis. In contrast, the presence of Francisella-like endosymbionts (FLEs) was detected in more than half of the D. reticulatus ticks (50.4%) and 0.8% of the I. ricinus ticks. The nucleotide sequences of the FLEs detected in D. reticulatus exhibited 100% homology with the nucleotide sequence of the FLE strain FDrH detected in Hungary in D. reticulatus. In conclusion, our results suggest a low contribution of D. reticulatus and I. ricinus ticks to the circulation of F. tularensis in eastern Poland. This finding, however, needs to be confirmed by further studies in other areas. Our study confirmed the common infection of D. reticulatus with Francisella-like endosymbionts (FLEs) of unknown pathogenic potential and revealed, for the first time, a low grade of infection of I. ricinus with FLEs. © 2015 Elsevier GmbH. All rights reserved.
Introduction Francisella tularensis (F. tularensis) is a fastidious, aerobic, gram-negative coccobacillus that affects vertebrates, especially lagomorphs and rodents. In humans, this coccobacillus causes tularemia, a potentially fatal multi-systemic zoonotic disease that occurs in the northern hemisphere, including North America, Europe, and Asia. The species F. tularensis is divided into three subspecies: subsp. Tularensis, which has high virulence and occurs predominantly in North America, subsp. Holarctica, which has mild virulence and occurs throughout the northern hemisphere, and subsp. Mediasiatica, which has mild virulence and occurs in Central Asia (Carvalho et al., 2014). Humans can become infected by F. tularensis by being bitten by hematophagous arthropods (ticks, mosquitos, and deer flies), by direct contact with infected animals (mostly hares and rabbits), by ingestion of contaminated food or water, and by inhalation of infected aerosols. The bacterium is highly infectious and is considered as a potential bioweapon (Carvalho et al., 2014; CDC, 2005;
∗ Corresponding author at: Department of Zoonoses, Institute of Rural Health, Jaczewskiego 2, 20-090 Lublin, Poland. Tel.: +48 817184555; fax: +48 817184555. E-mail address: [email protected] (A. Wójcik-Fatla). http://dx.doi.org/10.1016/j.ttbdis.2015.01.005 1877-959X/© 2015 Elsevier GmbH. All rights reserved.
Foley and Nieto, 2010; Zhang et al., 2008; Franke et al., 2010; Kaysser et al., 2008; Kugeler et al., 2005; Kreizinger et al., 2013). The transovarial transmission of F. tularensis in ticks is controversial and may depend on what subpopulations of F. tularensis and what tick species are present (Petersen et al., 2009). The principal tick vectors include species of the genera Amblyomma, Dermacentor, Haemaphysalis, Ixodes and Ornithodoros (Gordon et al., 1983). Many tick species are also hosts of bacteria that are closely related to F. tularensis, called Francisella-like endosymbionts (FLEs) (Dergousoff and Chilton, 2012). The pathogenic potential of FLEs remains unknown, although sequences homologous to the iglC and mglA genes of F. tularensis, which have been implicated in bacterial pathogenicity, have been detected in FLEs (MachadoFerreira et al., 2009). FLEs appear to replicate intracellularly, and they are transmitted transovarially. To date, there is no evidence of horizontal transmission through tick bites (Ivanov et al., 2011). FLEs are widely distributed, and a number of diverse FLEs have been reported in various tick genera on at least four continents (2011; Scoles, 2004). In Europe, thus far, FLEs have been isolated from Dermacentor reticulatus (D. reticulatus) in Hungary (Kreizinger et al., 2013; Sréter-Lancz et al., 2009), Portugal (De Carvalho et al., 2011), France (Michelet et al., 2013) and Germany (Gehringer et al., 2013) and from Hyalomma marginatum marginatum,
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males, and for nymphs the DNA concentrations ranged from 18 to 80 ng/l. Detection of Francisella spp. DNA in ticks by PCR
Fig. 1. Map of collection of ticks located in the Lublin province, eastern Poland (source of map http://www.polskainfo.pl). Locality A: Ostrów Lubelski; B: Parczew; C: Włodawa; D: Zwierzyniec.
Hyalomma aegyptium, Rhipicephalus sanguineus and D. reticulatus in Bulgaria (Ivanov et al., 2011). No recent reports are available on the occurrence of F. tularensis in potential arthropod vectors in Poland. To fill this gap, we examined samples of D. reticulatus and I. ricinus ticks collected from eastern Poland for the presence of F. tularensis; we also considered the potential for FLEs in the sampled ticks. Materials and methods Collection of ticks A total of 530 questing D. reticulatus ticks (273 females and 257 males) and 861 questing I. ricinus ticks (200 females, 179 males and 482 nymphs) were collected during the spring/summer season in 2011–2012 in 4 localities situated in the Lublin province (eastern Poland). D. reticulatus ticks were collected in 3 localities situated ˛ ´ on the Łeczy nsko-Włodawskie Lakeland: Ostrów Lubelski (locality ‘A’), Parczew (locality ‘B’), and Włodawa (locality ‘C’). I. ricinus ticks were collected in a forest inspectorate, Zwierzyniec (locality ‘D’), situated on the Roztocze Highland (Fig. 1). Ticks were collected by dragging a woolen flag over the lower vegetation and litter along the paths and edges of deciduous and mixed forests. DNA isolation from ticks Total DNA was isolated from the adult ticks separately and from nymphs in pools of 5 specimens (Rijpkema et al., 1996) by boiling in 0.7 M ammonium hydroxide. The concentration of DNA in the isolates was determined with the NanoDrop ND1000 Spectrophotometer (USA). For D. reticulatus, the determined DNA concentrations ranged from 520 to 672 for males and from 670 to 878 ng/l for females. For I. ricinus, the DNA concentrations ranged from 309 to 506 ng/l for females and from 175 to 328 ng/l for
For the 16S rRNA (rrs) and tul4 (encoding 17 kDa lipoprotein) genes, PCR assays were performed according to the methods of Michelet et al. (2013) with some modifications. A 50 l reaction volume was used and contained the following: 1 U Taq DNA polymerase (Qiagen, USA), 1× PCR buffer containing 15 mM MgCl2 , 2 mM dNTP (final concentration 0.2 mM) (Fermentas, Lithuania), 2.5 l 10 M each of primer (Eurogentec, Seraing, Belgium), 2 l of DNA and nuclease-free water (Applied Biosystems, USA). For the rrs and tul4 genes, the primers F11 and F5 (Forsman et al., 1994) and FT393 and FT642 (Long et al., 1993) were applied, respectively. The amplification was carried out in C1000 Thermal Cycler (BioRad, USA). After electrophoresis in a 1.5% agarose gel under standard conditions and staining with ethidium bromide solution (2 g/ml), the products of the 16S rRNA amplification were identified as a 1140 bp band. For the amplification products of tul4, electrophoresis was performed in 2% agarose gels, and a 248 bplong electrophoresis band was considered positive. The F. tularensis strain FSC043 was used as the positive control and was kindly provided by Prof. M. Kondrusik (Department of Infectious Diseases and Neuroinfection in Białystok, Poland). For the negative control, instead of using matrix DNA, we used nuclease-free water. In addition, positive specimens were examined using the lpnA gene, which encodes a region of a 17 kDA lipoprotein that is different from tul4. A 233-bp fragment of lpnA has been recommended as an appropriate marker for differentiating between F. tularensis and FLEs (Forestal et al., 2008; Escudero et al., 2008). The PCR was performed according to the method of Gehringer et al. (2013). After PCR, the samples that contained rrs, tul4 and lpnA genes were classified as positive. DNA sequencing DNA sequencing was performed with an ABI PRISM 310 Genetic Analyzer (Applied Biosystems, Inc., Foster City, CA, USA) using Abi Prism Big Dye Terminator v. 3.1. Cycle Sequencing Kits and Big Dye XTerminator Purification Kits (Applied Biosystems). The same primers as those used for the PCR of the rrs, tul4, lpnA genes were also used for sequencing. The resulting sequences were compared with sequences in the GenBank database using the BLAST server on the National Center for Biotechnology Information website (Bethesda, MD, USA). Statistical analysis The obtained results were analyzed by 2 test and Student’s ttest using the STATISTICA v. 6.0 package (Statsoft, Tulsa, OK, USA). A p value <0.05 was considered statistically significant. Results Only one female D. reticulatus tick out of 530 examined (0.2%) was infected with the F. tularensis subspecies holarctica according to the PCR results for the rrs gene (accession number CP007148). Unfortunately, the sequence analyses of tul4 and lpnA genes were not successful because of the poor quality of the PCR products (the bands on the agarose gel were too weak). In contrast, Francisella-like endosymbionts (FLEs) were detected in more than half of the D. reticulatus ticks examined (50.4%) (Table 1). FLE infection of ticks was significantly dependent on locality (p = 0.0083) and sex: females were infected significantly more often than males (p = 0.0034). None of the 861 I. ricinus ticks
267/530 (50.4%) 91/191 (47.6%) 0/191 (0)
1/530 (0.2%)
154/273 (56.4%) 113/257 (44.0%) 64/96 (66.7%) 27/95 (28.4%) 0/96 (0) 0/95 (0)
1/273 (0.4%) 0/257 (0)
Francisella-like endosymbionts (FLEs) Francisella tularensis ssp. holarctica Francisella-like endosymbionts (FLEs) Francisella tularensis ssp. holarctica
74/191 (38.7%) 102/148 (68.9%) 1/148 (0.7%) Total
0/191 (0)
32/96 (33.3%) 42/95 (44.2%) 58/81 (71.6%) 44/67 (65.7%) 1/81 (1.2%) 0/67 (0) Females Males
0/96 (0) 0/95 (0)
Francisella-like endosymbionts (FLEs) Francisella tularensis ssp. holarctica Francisella tularensis ssp. holarctica
Francisella-like endosymbionts (FLEs)
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Table 2 Prevalence of Francisella tularensis and Francisella-like endosymbionts (FLEs) determined with the genes tul4 and rrs in Ixodes ricinus ticks found on the territory of the Lublin province (eastern Poland). Locality/species
Locality ‘D’ Infected/examined (percent)
Stage
Francisella tularensis ssp. holarctica
Francisella-like endosymbionts (FLEs)
Females Males Nymphs
0/200 (0) 0/179 (0) 0/482 (0)
5/200 (2.5%) 2/179 (1.1%) 0/482 (0)
Total
0/861 (0)
7/861 (0.8%)
were infected with F. tularensis. The presence of Francisella-like endosymbionts (FLEs) was detected in 0.8% of the I. ricinus ticks (Table 2). FLE infection of I. ricinus ticks was significantly dependent on stage. None of the nymphs were infected with FLEs, and this result was significantly lower than that observed in the female (p = 0.0005) and male (p = 0.0214) ticks. Sequence analysis of 30 randomly selected samples of D. reticulatus that were positive for FLEs and seven randomly selected samples of I. ricinus that were positive for FLEs showed 100% homology with the nucleotide sequence of the tul4 gene (GenBank accession no. EU126640) from an FLE detected previously in D. reticulatus in Hungary. The sequence analysis also showed 100% homology of the lpnA gene sequences of the samples with the FLEs with accession numbers FN686808–FN686813, FN686815, FN686816, HM629449, and KJ477082 and 100% homology of the rrs gene sequence of the samples with the FLEs with accession numbers JX561116, JQ942365, HQ705173 and EU234535. Discussion
Stage
Total Infected/examined (percent) Locality ‘C’ Infected/examined (percent) Locality ‘B’ Infected/examined (percent) Locality ‘A’ Infected/examined (percent) Locality/species
Table 1 Prevalence of Francisella tularensis and Francisella-like endosymbionts (FLEs) determined with the genes tul4 and rrs in Dermacentor reticulatus ticks found on the territory of the Lublin province (eastern Poland).
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In this study, for the first time, we report the occurrence of F. tularensis subsp. holarctica and Francisella-like endosymbionts (FLEs) in ticks from Poland. The epidemiological situation of tularemia in Poland is not fully known. The first human case of tularemia was described in 1949, and from 1949 to 2009 a total of 614 cases were reported and were largely attributed to contact with infected hares (Kłapec´ and Cholewa, 2011). In 2012 and 2013, only 6 and 8 cases were reported, respectively (morbidity for 100,000 equal to 0.02) (PZH, 2013). Pancewicz et al. (2004) found a low prevalence of reactions to the F. tularensis antigen serologically among forestry workers (2.06%), casting doubt on the role of the I. ricinus tick in the epidemiology of tularemia in Poland. The results of a study by Rastawicki et al. (2006) showed that antibodies to F. tularensis were detected slightly more often in healthy forest workers than in healthy blood donors, but this difference was not statistically significant. Until now, nothing has been reported on the incidence of tularemia in humans and animals in eastern Poland, which was the location of tick sampling in the present study. The D. reticulatus ticks collected in eastern Poland were infected with the zoonotic bacterium F. tularensis subsp. holarctica at a low rate of 0.2%. This is in accordance with the results of a study in Hungary by Kreizinger et al. (2013), which showed a prevalence of F. tularensis in D. reticulatus ticks equal to 0.27%. Our results are also in accordance with those of Gehringer et al. (2013) from Germany, Bonnet et al. (2013) from France, and Reye et al. (2013) from Belarus, none of whom found F. tularensis subsp. holarctica in D. reticulatus. Our observed rate of infection was lower than that noted by De Carvalho et al. (2007) in Portugal (1.1%). It was also lower than those reported earlier from natural foci of tularemia in the Czech Republic and Austria by Hubálek et al. (1996, 1997, 1998), who found a prevalence of virulent strains of F. tularensis in D. reticulatus equal to 2.3–2.6% and was lower than that reported
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from a natural focus of tularemia in western Slovakia by Gurycová et al. (1995), who found a prevalence ranging from 0.5 to 2%. Hubálek et al. (1997) have the opinion that D. reticulatus plays an important role as a vector of tularemia in endemic areas. In this context, our results suggest that for the examined area of eastern Poland, F. tularensis is not circulating among D. reticulatus ticks to any great extent and its role as a vector of tularemia in this area is unknown. In contrast to F. tularensis, the prevalence of FLEs was high (50.4%). This confirms the reports from other European countries (Kreizinger et al., 2013; Sréter-Lancz et al., 2009; De Carvalho et al., 2011; Michelet et al., 2013; Gehringer et al., 2013) that D. reticulatus is a suitable host for these bacteria. Our result is similar to that reported from Germany (57%) (Gehringer et al., 2013) higher than those reported from Hungary (1.2–3%) (Kreizinger et al., 2013; Sréter-Lancz et al., 2009) and Portugal (39%) (De Carvalho et al., 2011), and lower than that reported from France (79%) (Michelet et al., 2013). It should be mentioned that some of these comparisons might not be accurate because of the differences in the PCR methods used by the individual researchers and the differences in the numbers of ticks tested. None of the I. ricinus ticks examined by us were infected with F. tularensis, and this observation is in agreement with those reported by Sréter-Lancz et al. (2009), Egyed et al. (2012), and Kreizinger et al. (2013) from Hungary, Bonnet et al. (2013) from France, Reye et al. (2010) from Luxembourg and Sanogo et al. (2003) from Italy, who also did not find this bacterium in I. ricinus. Low grade infection rates of I. ricinus with F. tularensis were reported from the Czech Republic (0.2%) (Hubálek et al., 1996), Slovakia (0.3%) (Gurycová et al., 1995), France (0.8%) (Reis et al., 2011), Central Germany (1.2–1.6%) (Franke et al., 2010), Belarus (1.4%) (Reye et al., 2013), and Serbia (3.8%) (Milutinovic´ et al., 2008). A distinctly higher infection rate of I. ricinus with the F. tularensis subsp. holarctica was found by Gehringer et al. (2013) in a territory of endemic focus of tularemia in Baden-Wuerttemberg, Germany. In contrast, not FLEs were detected in 916 I. ricinus ticks. Similarly, in Hungary, of 3222 I. ricinus ticks none were found to be infected with FLEs (Kreizinger et al., 2013). Although the same genetic markers were used for all studies, the results of Kreizinger et al. contrast those of our study, which only showed FLEs in I. ricinus. To the best of our knowledge, this report is the first to describe the presence of Francisella-like endosymbionts (FLEs) in adult I. ricinus ticks. In conclusion, our results suggest a low contribution of D. reticulatus and I. ricinus ticks to the circulation of F. tularensis in eastern Poland. However, this finding needs to be confirmed by further studies in other areas, especially considering the possible occurrence of specific microfoci reported by Goethert and Telford (2009). Our study confirmed the common infection of D. reticulatus with Francisella-like endosymbionts (FLEs) of unknown pathogenic potential and revealed, for the first time the low grade infection of I. ricinus with FLEs.
Acknowledgment This study was supported by the National Science Centre (Grant: N N40404267640).
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