Anaplasma phagocytophilum, Babesia microti, Borrelia burgdorferi sensu lato, and Toxoplasma gondii in Ixodes ricinus (Acari, Ixodida) ticks collected from Slowinski National Park (Northern Poland).

Abstract

Ticks are important vectors of many pathogens, including Borrelia burgdorferi sensu lato, Anaplasma phagocytophilum, Babesia microti, and probably Toxoplasma gondii. These pathogens are ethiological agents of many diseases, such as Lyme borreliosis, human granulocytic anaplasmosis, babesiosis, and toxoplasmosis (Stańczak et al. 2004, Sroka et al. 2009). In Europe and Poland, the main vector of these pathogens is Ixodes ricinus (Nowak-Chmura 2013). In Poland, this tick is active from early spring to late autumn and it can feed on terrestrial vertebrates including humans (Bartosik et al. 2011, Nowak-Chmura 2013). Slowinski National Park (SNP) is situated in northern Poland and it is one of the two seaside national parks in Poland (Figure 1). The park is frequently visited by tourists because of its natural beauty but its ticks fauna has not yet been thoroughly investigated (Semla et al. 2013). This situation can be dangerous for tourists and the local community who can suffer from tick bites and transmitted diseases. Tick-borne diseases in Poland are a serious problem with most cases having been observed in the provinces of northern Poland (Szulzyk and Flisiak 2012). Lyme disease is particularly threatening to people who are professionally connected with the tick's natural environment (people working in forests, national and landscape parks) (Bartosik et al. 2011). Most likely, not all cases are correctly identified, and not all are reported to the registry. Therefore, it seems necessary to conduct research on the prevalence and epidemiology of the ticks in any given area. Therefore, the aim of the study was to evaluate the role of I. ricinius nymphs in the transmission of B. burgdorferi s.l., A. phagocytophilum, B. microti, and T. gondii in the selected areas of SNP and to analyze the risk of exposure to these pathogens. Furthermore, the results of these studies may help deepen public awareness concerning the dangers connected with tick attacks in the studied areas of SNP. The ticks were collected by flagging in the selected areas of SNP during the summer of 2013 and identified using the key by Nowak-Chmura (2013). Generally, an area of 1000 m2 was flagged. The DNA was isolated by the ammonia method. The pathogens in ticks were detected by PCR and nested PCR. To detect B. burgdorferi s.l., a pair of primers Fla1/Fla2 specific to the flagelline gene was used (Wójcik-Fatla et al. 2009). In turn, to detect A. phagocytophilum, the two pairs of primers ge3a/ge10r and ge9f/ge2r specific to the 16S rDNA gene were used (Massung et al. 1998). To detect B. microti, the two pair of primers Bab1/Bab4 and Bab2/Bab3 specific to the 18S rRNA coding gene for a small ribosome subunit were used (Wójcik-Fatla et al. 2009). The two pairs of primers Pm1S1/Pm1AS1 and Pm1S2/Pm1AS2 specific to B1 gene were used to detect T. gondii (Sroka et al. 2009). The amplification products were separated electrophoretically in 2% ethidium bromide stained agarose gels and they were visualized and photographed in an analyser Omega 10 (UltraLum, U.S.A.). The statistical analysis was performed using CSS-Statistica for Windows ver. 10. Statistical significance was declared at a p value of less than 0.05. Results were analyzed using the χ2 test. A total of 120 I. ricinus ticks was collected, including 112 nymphs and eight males. Only nymphs were analyzed for the presence of B. burgdorferi s.l., A. phagocytophilum, B. microti, and T. gondii. The molecular studies showed that only 0.9% of ticks were infected with A. phagocytophilum whereas 15.2% were infected with B. microti. The protozoans T. gondii appeared in 4.5% of the examined nymphs (Table 1). The rickettsiae A. phagocytophilum were present only in 3.6% of the ticks collected from Smoldzino, whereas the protozoan T. gondii was present only in 14.7% of the nymphs collected from Czolpino (Table 1). The piroplasms B. microti were significantly more frequent in the nymphs collected from Czolpino (32.3%) than in the nymphs from Smoldzino (10.7%) and Lupawa Valley (6.0%) (χ2 = 13.06, p < 0.0005 and χ2=21.96, p ≤ 0.00001, respectively). The difference between Smoldzino and Lupawa Valley was not statistically significant (χ2 = 1.61; p = 0.20). In the area of Czolpino the co-existence of three pathogens, A. phagocytophilum, B. microti, and T. gondii, in one nymph was shown, whereas the co-existence of B. microti and T. gondii in one nymph collected from Lupawa Valley has been shown. The co-infection of A. phagocytophilum and T. gondii appeared in one nymph from Smoldzino. B. burgdorferi s.l. was not detected in any of the examined material (Table 1). Human infections with Babesia species, in particular B. microti, are tick-borne illnesses that are being recognized with increased frequency. The clinical presentation of babesiosis ranges from an asymptomatic form to a life-threatening infection with severe haemolysis (Poisnel et al. 2013). Our studies showed a high percentage of nymphs infected with B. microti in the territory of SNP. The received results showed a 50% higher level of I. ricinus infected with these protozoans than in other regions of Poland where only 2–7% of ticks were infected with B. microti (Stańczak et. al 2004, Skotarczak et al. 2008, Wójcik-Fatla et al. 2009, Asman et al. 2014). The prevalence of ticks infected with A. phagocytophilum ranges in Europe from 0.4–66.7% (Blanco and Oteo 2002). The presence of A. phagocytophilum in I. ricinus in Poland is also different and it ranges from 4.94% in eastern Poland (Wójcik-Fatla et al. 2009) to 76.7% in the territory of the Niepołomice Forest (Asman et al. 2013). The highest rate of infection was found in female ticks (45.7%), whereas for males and nymphs the rates of infection constituted only 4.5% and 0.9%, respectively (Tomasiewicz et al. 2004). The results are similar to those in eastern Poland and may confirm the low presence of this pathogen in nymphs. The presence of B. burgdorferi s.l. in I. ricinus nymphs ranged from 2% in eastern Poland (Cisak et al. 2012) up to 62% in some regions of southern Poland (Asman et al. 2012). In contrast to the previous studies conducted in different regions of Poland, this pathogen did not appear in examined nymphs collected from the selected areas of SNP. The absence of this pathogen in the collected material could be due to the number of collected and examined ticks as well as the lower frequency of occurrence of this spirochete in ticks in this region of Poland. Knowledge of the presence of T. gondii in I. ricinus ticks in Poland is still very poor. The percentage of infected ticks in Poland varies between 6.1%–64.9%. In all these studies, the most infected stage of I. ricinus were females, whereas the percentage of infected nymphs varies between 0–5.6% (Sroka et al. 2003, 2008, 2009, Asman et al. 2015). The results may confirm the low level of infection with T. gondii in I. ricinus nymphs. Skotarczak et al. (2008) in northwestern Poland and Wójcik-Fatla et al. (2009) in eastern Poland showed similarity to the co-existence of rickettsiae A. phagocytophilum and protozoans Babesia. The higher co-existence of these two pathogens was showed by Stańczak et al. (2004) in northern Poland, and Asman et al. (2014) in southern Poland. There, co-existence of B. burgdorferi s.l. and T. gondii is known, as is B. microti and T. gondii in I. ricinus (Sroka et al. 2009, Asman et al. 2015). In these studies, the co-existence of these two protozoans in ticks has been confirmed. Additionally, the co-existence of A. phagocytophilum and T. gondii was found. The co-existence of three or more pathogens in one tick is usually observed in a low percentage of ticks (Wójcik-Fatla et al. 2009). The obtained results may confirm this fact and additionally it may indicate the possibility of co-existence of A. phagocytophilum, B. microti, and T. gondii in a single specimen of I. ricinus. In conclusion, the detection of infection with B. microti, A. phagocytophilum, and T. gondii in I. ricinus nymphs elucidated their role in maintaining these pathogens in the studied areas and environments of SNP. The significantly higher incidence of the piroplasms of the genus Babesia in nymphs in the SNP demonstrated that nymphs of I. ricinus are potential sources of this tick-borne disease in humans in the examined area of Poland. The presently reported detection of B. microti in 32.3% of nymphs collected from Czolpino, in 10.7% of the nymphs from Smoldzino, and in 6.0% of the nymphs from Lupawa Valley indicates their possible role in the epidemiology of babesiosis in these locations. Moreover, the presence of T. gondii in 14.7% of the ticks from Czolpino may indicate that I. ricinus can probably be a potential vector for this dangerous protozoan in these locations in the territory of the SNP. A potential risk of exposure of humans, including tourists, to nymphs of I. ricinus infected with B. microti and T. gondii in the examined locations of the SNP has been stated, especially in Czolpino. On the other hand, the highest risk of exposure to nymphs infected with A. phagocytophilum was detected in Smoldzino.