Molecular detection of tick-borne pathogens in ticks collected from pets in selected mountainous areas of Tatra County (Tatra Mountains, Poland)

The mountainous and foothill areas, in which the city of Zakopane, the capital of Tatra County, is located are characterized by continuous weather changes, lower air temperature, persistent snow cover, and poorer vegetation than in the lowlands. Ixodes ricinus and Ixodes hexagonus are vectors of tick-borne diseases and play an important role in the persistence of tick-borne diseases. The aim of the study was to determine the risk of exposure of domestic cats and dogs to the attacks of Ixodid ticks, to tick-borne infections with Borrelia burgdorferi sensu lato, Anaplasma phagocytophilum, Babesia microti and Toxoplasma gondii in the city of Zakopane and the surrounding area. In 2017–2018 ticks were collected from a total of 10 domestic cats and 88 domestic dogs. Selected pathogens of tick-borne diseases were detected by PCR and nested PCR. The study material contained 119 I. ricinus and 36 I. hexagonus. The molecular examinations showed the presence of A. phagocytophilum in 3.8%, B. microti in 24.5% and T. gondii in 4.5% of the all ticks. In addition, in the study area, there is a high potential risk of tick-borne infection by B. microti, and a low potential risk of exposure to A. phagocytophilum and T. gondii infection.

Scientific RepoRtS | (2020) 10:15865 | https://doi.org/10.1038/s41598-020-72981-w www.nature.com/scientificreports/ Due to the great medical and veterinary importance of ticks and close contact between human and domestic animals, an attempt has been made to determine the exposure of cats and dogs to tick infection, as well as to a potential tick-borne infection with B. burgdorferi s. l. and A. phagocytophilum, as well as to the infection of Babesia microti and Toxoplasma gondii in the selected areas of Tatra County. The approach of gathering data on the distribution of (zoonotic) vector-borne diseases through a veterinary survey is consistent with the 'One Health' concept 10 .

Materials and methods
Zakopane, is the highest located city in Poland (49° 18′ N; 19° 57′ E), situated in the Podtatrzanski Trench, between the Tatra Mountains and the Beskids, in one of the largest mountain ranges in Europe-the Carpathian Mountains. The urban part stretches from 750 to 1000 m a.s.l., the administrative boundaries of the city contain a part of the Tatra Mountains with the highest point-the peak of Swinica (2301 m a.s.l.), as well as Gubalowka (1120 m a.s.l.), and the central point of Zakopane is located at an altitude of 838 m a.s.l.
The research material was obtained from domestic cats and dogs in 2017-2018 from March to September in cooperation with the veterinary clinic in Zakopane. The ticks were collected from animals using tweezers and placed in tubes with 70% ethyl alcohol. In addition, after the collection, an original form was completed with the following information: date of collection, animal breed, sex, age and the city of collection. Then, individual ticks were determined for genus, species and developmental stage. The keys by Siuda 1 and Nowak-Chmura 7 were used to identify the ticks. Next, molecular testing for the presence of selected pathogens was performed. DNA was isolated from 155 ticks using the ammonium hydroxide method 11 . Then, the concentration was measured spectrophotometrically at the wave length of 260/280 nm. The pathogens in the material were detected by PCR and nested PCR. For detection of B. burgdorferi s.l., Maximo DFS-Plus polymerase (GeneOn, Germany) and flagellin gene specific primers were used 12 . Taq DNA polymerase (EURx, Poland) and two pairs of specific primers for the 16S rRNA gene were applied to detect A. phagocytophilum 13 . The protozoa of B. microti and T. gondii were detected with two pairs of specific primers for the 18S rRNA gene and Maximo DFS-Plus polymerase (GeneOn, Germany) and for the B1 gene with the use of Taq DNA polymerase (EURx, Poland) 14,15 . The amplification and re-amplification products were separated electrophoretically on 2% ethidium bromide stained agarose gels. Then, the gels were visualized in ultraviolet light. The following reaction products were treated as positive: 482 base pairs [bp] for B. burgdorferi s. l., 932 bp and 546 bp for A. phagocytophilum, 238 bp and 154 bp for B. microti and 531 bp for T. gondii. Statistical analysis was performed using CSS-Statistica for Windows 10. Statistical significance was accepted at a p value of less than 0.05. The results were analysed using an χ 2 tests. ethical approval. We declare that all testing methods have been carried out in accordance with the relevant guidelines and regulations. We declare that all experimental protocols have been approved by the Medical University of Silesia in Katowice and Pedagogical University in Cracow. informed consent. Each dog and cat owner has agreed to collect material (ticks) from their animals and give informed consent to publish the results of the collected material.

Results
A total of 155 ticks were collected from 10 domestic cats and 88 domestic dogs. I. ricinus was the dominant tick species, with 119 females collected from 90 animals, including 8 cats and 82 dogs. 36 I. hexagonus ticks were also collected, including 15 larvae, 13 nymphs and 8 females. The presence of this tick species has been demonstrated in 8 animals, including 2 cats and 6 dogs. The infestation by I. ricinus usually occurred in May, while it was least often reported in March and September (Fig. 1).
I. hexagonus species usually invaded in July, and it was least frequently found in March and May (Fig. 2). In total, the pathogens were found in 26.0% of I. ricinus individuals and in 50.0% of I. hexagonus ticks. This difference was statistically significant (Yates corrected χ 2 = 11.23; p = 0.0008). A. phagocytophilum was found only in 3.4% of female I. ricinus ticks. On the other hand, protozoa of B. microti and T. gondii were reported in both tick species. B. microti was reported in a much higher percentage of I. ricinus and I. hexagonus individuals than T. gondii (Table 1). B. burgdorferi s. l. were not shown in the all study material. In addition, the coexistence of A. phagocytophilum and B. microti, as well as A. phagocytophilum and T. gondii was demonstrated in I. ricinus females (Table 1). In general, all ticks collected from cats and dogs were statistically significantly more often infected with B. microti than with T. gondii and A. phagocytophilum (Yates corrected χ 2 ; p ≤ 0.00001, in all cases).
Mainly larvae of I. hexagonus contained B. microti (46.7%), whereas T. gondii was found in a similar percentage in both juvenile forms ( Table 2). The difference was statistically significant (Yates corrected χ 2 = 38.58 and 36.21 for larvae and nymphs, respectively; p ≤ 0.00001, in both cases).
Generally, I. hexagonus collected from cats and dogs were statistically significantly more often infected with B. microti than with T. gondii (Yates corrected χ 2 = 10.96; p = 0.0009).
Babesia microti was found in 28.1% of the ticks collected from dogs. A. phagocytophilum and T. gondii were found in 3.9% of the I. ricinus females (Table 3). This difference was statistically significant (Yates corrected χ 2 = 19.68; p ≤ 0.00001). In addition, single cases of A. phagocytophilum and B. microti as well as A. phagocytophilum and T. gondii were reported in the ticks collected from dogs ( Table 3).
As for I. hexagonus ticks collected from cats, B. microti was shown in 54% of the nymphs and in 33.3% of the females. It should be emphasized that this difference was statistically significant (Yates corrected χ 2 = 8.14; p = 0.0043). T. gondii was demonstrated in 7.1% of the larvae and in 7.7% of the nymphs of this tick species. Generally, hedgehog ticks collected from cats were statistically significantly more often infected with B. microti than with T. gondii (Yates corrected χ 2 = 12.79; p = 0.0003).    33 in Tarnowskie Góry County. The researchers showed the presence of B. microti in 42.6% of the individuals, while T. gondii was in 98% of the ticks. In addition, these protozoa have been demonstrated in ticks collected from both cats and dogs 33 . Contrary to the studies conducted by Asman et al. 33 , B. microti and T. gondii have been shown in I. ricinus ticks collected only from dogs, and in I. hexagonus species collected only from cats. However, like in the analyses of ticks conducted in Tarnowskie Góry County, both these protozoan species were found mainly in I. ricinus females. This confirms the thesis that, apart from the nymph, this developmental stage is the main epidemiological threat for protozoa 33 . Ticks may play a large role in the transmission of T. gondii, but this requires further research.
Anaplasma phagocytophilum was found in a much smaller percentage of I. ricinus individuals and was not reported in I. hexagonus. The values are much lower than those obtained by Król et al. 8 who demonstrated the   32 .
It is commonly known that ticks can be vectors and/or reservoirs of more than one pathogen. There are cases of co-occurrence of two or three pathogens in I. ricinus ticks collected from vegetation, but the percentage of such ticks in the population is very low 12,[35][36][37] . Also, there are cases of such coinfection in ticks collected from domestic animals 8,33 . The research conducted in Tarnowskie Góry County by Asman et al. 33 showed the co-occurrence of B. microti and T. gondii in more than 40% of I. ricinus ticks collected from cats and dogs. Moreover, co-infection was reported mainly in female I. ricinus ticks collected from dogs. On the other hand, Król et al. 8 demonstrated the coexistence of 2 or even 3 pathogens in a single I. ricinus tick, with A. phagocytophilum and Rickettsia spp. most frequently found in co-infection. The ticks collected from dogs in Tatra County showed coexistence of A. phagocytophilum and B. microti as well as A. phagocytophilum and T. gondii in only two I. ricinus females. However, coexistence of these pathogens was not observed in the ticks collected from cats, which may result from a small number of ticks collected from these animals.

conclusions
The research indicates that potentially unfavourable environmental conditions for ticks in Tatra County do not prevent domestic cats and dogs from a high risk of exposure to the infestation by I. ricinus and I. hexagonus ticks. In addition, the study revealed a potentially high risk of tick-borne infection of B. microti and a low risk of exposure to a tick-borne A. phagocytophilum infection and T. gondii invasion in the study area. Moreover, the results show possible coexistence of A. phagocytophilum and both B. microti and T. gondii in I. ricinus species. However, it cannot be excluded the possibility that some individuals may have been pathogen-positive because of feeding on an infected (asymptomatic) animal. The lack of Borrelia burgdorferi s. l. in the material may be due to a generally low percentage of ticks infected with this bacterium in the study area, and this may result from the geographical location.