Wild-living animals play a significant role in epidemiology of zoonoses; they constitute the main source of pathogens dangerous for humans and domestic animals. Number of zoonotic agents carried by wild-living animals increases and still the threat they pose is not well known, particularly for humans with direct contact to animals. The scope of danger may vary and it depends not only on the source of infection but also on transmission routes (Artois 2003; Jones et al. 2008). The presence of vectors in the environment (e.g. ticks) is correlated with the existence of ecological niches inhabited by the hosts and tick-specific environmental conditions (temperature and humidity levels) (Daszak et al. 2000). Ticks are among the most common zoonosis vectors; tick-borne diseases (TBDs) are a significant group of diseases impacting public health.
Some of the zoonosis-related threats are the diseases caused by
In order to monitor the presence of
The goal of this study was to determine the occurrence of
The wild animals to be sampled were hunted in the years 2016–2017 in the areas, which included the hunting of the following clubs “Cyraneczka” – Drawsko Pomorskie; “Żbik” – Cieszyno near Złocieniec; “Bażant” – Stawno near Złocieniec (Fig. 1). For testing, the samples were collected from internal organs (liver, spleen, heart and lungs). In total, 928 tissue samples were collected from 232 animals from the following species: red deer (140 animals), roe deer (40), and boar (52). The samples, which consisted of mixture of tissues (5–10 g) from four organs examined were suspended in saline solution and homogenized (rotor-stator homogenizer – SHM1, Stuart). Subsequently, 1 ml of the homogenizate was transferred to a new 2 ml test tube and centrifuged for 1 min at room temperature at 6300 rpm. The supernatant (1000 µl) was transferred to a new test tube and centrifuged again for 3 minutes at room temperature at 15 000 rpm. The pellet was resuspended in a lysis buffer, then 2 µl RNase and 10 µl proteinase K were added and incubated for 18 hours at 56°C. Subsequent stages were carried out according to the protocol of GeneMatrix Tissue&Bacterial DNA Purification Kit (EURx Ltd., Poland).
The research also included 1551 ticks collected from five testing sites that were forested areas of Drawsko military training ground (Konotop Encampment, Oleszno and Karwice, Drawa and Konotop Lake) from April to May 2017 (Fig. 1). These ticks were collected by the flagging-dragging method. Tick species were identified using taxonomic keys; the species collected was found to be
80 pooled tick samples (pools) were obtained. Among them, 72 pools contained 20 tick imagos or nymphs (10 pools – ♂, 12 pools – ♀, 50 pools – N), 3 pools contained 19 ticks (1 pool – ♂, 1 pool – ♀, 1 pool – N), 1 pool contained 17 ticks – ♀, 1 pool contained 13 ticks – ♂, 1 pool contained 12 ticks – ♀, 1 pool contained 7 ticks – N, 1 pool contained 5 ticks – N. In Drawa location pools were as follows: 211 (♀ – 2 pools/20 individuals, 1 pool/19 individuals; ♂ – 2 pools/20 individuals, 1 pool/12 individuals; N – 5 pools/20 individuals), in Karwice location the following samples were obtained: 460 (♀ – 5 pools/20 individuals; ♂ – 4 pools/20 individuals; N – 14 pools/20 individuals), in Lake Konotop location: 377 (♀ – 4 pools/20 individuals, 1 pool/13 individuals; ♂ – 3 pools/20 individuals, 1 pool/19 individuals; N – 10 pools/20 individuals, 1 pool/5 individuals), in Konotop location: 117 (♀ – 1 pool/17 individuals; ♂ – 1 pool/20 individuals; N – 4 pools/20 individuals), in Oleszno location: 386 (♀ – 1 pool/20 individuals, 1 pool/7 individuals; ♂ – 1 pool/19 individual, N – 17 pools/20 individuals) were collected, respectively. The pooled tick samples were placed in 2 ml test tubes with 300 µl of ethanol (70%) inside and left for 15 minutes (stirred several times). The alcohol was removed, and the ticks were rinsed with deionized water (300 µl). The residues of water were removed with blotting paper and the samples were placed in liquid nitrogen for 10 minutes. The ticks were then homogenized (mechanically, in a mortar) (Halos et al. 2004; Rodriguez et al. 2014; Jose et al. 2017). Following that, 1 ml of deionized water was added, and the samples were frozen at –80°C for further analyses. To isolate the genetic material, 200 µl of the homogenized liquid was used. The material was centrifuged at 15 000 rpm for 3 minutes at room temperature. The pellet was resuspended in lysis buffer LyseT, 2 µl RNase and 10 µl proteinase K were added and the material was incubated for 12 hours at the temperature of 56°C. Subsequent stages were carried out according to the protocol by GeneMatrix Tissue&Bacterial DNA Purification Kit (EURx Ltd., Poland).
Screening tests were performed by the real-time PCR method using a
The oligonucleotides used in real-time PCR.
Forward primer | 5’-GTCTTAAGGTGGGCTGCGT G-3’ | 5’-AACAATGGCACCTAGTAAT ATTTCTGG-3’ | 5’-ATTACAATGGCAGGCTCC AGA-3’ |
Reverse primer | 5’-CCCCGAATCTCATTGATC AGC-3’ | 5’-CCACCAAAGAACCATGTT AAACC-3’ | 5’-TGCCCAAGTTTTATCGTTC TTCT-3’ |
Probe | 5’-FAM-AGCGAACCATTGGTATC GGACGTTT-TAMRA-TATGG-Pho-3’ | 5’-FAM-TGGCAGAGCGGGTACT AACATGATTGGT-TAMRA-3’ | 5’-FAM TCCTAAGTGCCATGAT ACAAGCTTCCCAATTACTAAG-BHQ1-3’ |
The oligonucleotides were synthesized by Genomed S.A. (Poland). The reactions for both pathogens were conducted using LightCycler 2.0 instrument (Roche, Germany) according to the following thermal profile: initial denaturation at 95°C for 10 minutes; 40 cycles (95°C for 15 seconds, 60°C for 30 seconds); 40°C for 30 seconds. DNA extracted from
The prevalence of infected ticks in pools was analyzed based on the number of individuals. Statistical analyses to calculate estimated prevalence for fixed and variable pool sizes were performed with EpiTools (
Q fever research in Poland are based on immunological status of both domestic and wild-living animals (Niemczuk et al. 2011). Only few studies involved molecular analyses of clinical or environmental material (arthropods) (Tylewska et al. 1996; Szymańska et al. 2013; Bielawska-Drózd et al. 2014; Bielawska-Drózd et al. 2016). Seven wild-living animals (three boars, three stags and one roe deer) were found positive for
In this study any characteristic sequences were detected in testing for the presence of
Epidemiological situation of Q fever and tularemia in Poland as well as worldwide seems to be stable. Although Q fever and tularemia outbreaks have been registered almost all around the world, the numbers of infections are still low, but Scandinavian countries, Hungary and Czech Republic in relation to tularemia (Bielawska-Drózd et al. 2013; ECDC 2016). Most outbreaks of tularemia and Q-fever have been in wildlife species. In Germany between 2002 and 2016, 10 clusters of tularemia were reported. A serological study in various wildlife species in Brandenburg revelated a total of 101/1353 positive sera (7.5%) of foxes, raccoon dogs, and wild boars (Faber et al. 2018). The reports from Germany between 1992 and 2012 showed that 2.4% of dead wild European rabbits were positive for
The estimated prevalence of
The estimated prevalence of positive ticks pools for
Site | Number of pools | PoolSize | Number of positive pools for | Number of positive pools for | Number of individuals | Total number of individuals | Estimated prevalence (%)1 | Estimated prevalence (%)1 |
---|---|---|---|---|---|---|---|---|
Drawa | 9 | 20 | 1 | 0 | 59 | 211 | 0.5% | 0.49% |
1 | 19 | 0 | 0 | 52 | ||||
1 | 12 | 0 | 1 | 100 | ||||
Karwice | 23 | 20 | 2 | 0 | 460 | 460 | 0.45% | 0 |
Lake Konotop | 17 | 20 | 8 | 0 | 340 | 377 | 3.1% | 0 |
1 | 19 | 0 | 0 | 19 | ||||
1 | 13 | 0 | 0 | 13 | ||||
1 | 5 | 1 | 0 | 5 | ||||
Konotop | 5 | 20 | 2 | 0 | 100 | 117 | 3.45% | 0 |
1 | 17 | 1 | 0 | 17 | ||||
Oleszno | 18 | 20 | 2 | 0 | 360 | 386 | 0.55% | 0 |
1 | 19 | 0 | 0 | 19 | ||||
1 | 7 | 0 | 0 | 7 |
The results obtained in this work correlate with the data found by other researchers. However, despite the results did not present an increase in the proportion of the ticks infected, it is worth to continue studies by Formińska et al. (2015) and Chmielewski et al. (2010) and to examine more numerous tick species and biological agents that can exist in arthropods and constitute a significant threat to human health and lives. It is also advisable to extend the scope of research to include other wild-living animal species as potential reservoirs of zoonotic pathogens.
Additionally, this research demonstrates the useful molecular tool for the detection of