Entomopathogenic Fungi in Forest Habitats of Ixodes ricinus.

(1) Background: In addition to the microclimate, host availability, and tick microbiota, soil environmental microorganisms can affect tick populations. This study aimed to (1) determine the presence and diversity of entomopathogenic fungi (EF) in forests, where ticks are abundant, and (2) estimate the effectiveness of the isolated EF strains against Ixodes ricinus. (2) Methods: EF were isolated using the trap insect method from soil collected from tick sites. A bioassay was used to estimate the effectiveness of EF against ticks. (3) Results: The presence of EF was found in all tested forest habitat types. A total of 53 strains belonging to the genera Metarhizium, Beauveria, and Isaria were isolated. All the six strains subjected to the bioassay showed potential efficacy against both adult and nymphal stages of I. ricinus; however, the strains differed in their effectiveness. The most effective isolate against I. ricinus was the soil environmental strain of Metarhizium anisopliae. (4) Conclusion: The study indicates that tick habitats can be the source of entomopathogenic fungi, which have a lethal effect on ticks, as demonstrated in preliminary laboratory tests with I. ricinus. However, for practical use, extensive field tests and further research on application methods and long-term effects are necessary to develop effective and sustainable tick management strategies.

Mosquitoes (Diptera: Culicidae) of Poland: An Update of Species Diversity and Current Challenges.

This article presents the current state of knowledge of mosquito species (Diptera: Culicidae) occurring in Poland. In comparison to the most recently published checklists (1999 and 2007), which listed 47 mosquito species, four species (Aedes japonicus, Anopheles daciae, Anopheles hyrcanus, and Anopheles petragnani) are added to the Polish fauna. Our new checklist of Polish mosquito fauna includes 51 species of mosquitoes from five genera: Aedes (30), Anopheles (8), Coquillettidia (1), Culiseta (7), and Culex (5). Aspects of the ecology and biology of the Polish mosquito fauna, with particular emphasis on newly recorded species, are discussed.

Identification of New Microfoci and Genetic Characterization of Tick-Borne Encephalitis Virus Isolates from Eastern Germany and Western Poland.

(1) Background: Tick-borne encephalitis (TBE) is the most important tick-borne viral disease in Eurasia, although effective vaccines are available. Caused by the tick-borne encephalitis virus (TBEV, syn. Orthoflavivirus encephalitidis), in Europe, it is transmitted by ticks like Ixodes ricinus and Dermacentor reticulatus. TBEV circulates in natural foci, making it endemic to specific regions, such as southern Germany and northeastern Poland. Our study aimed to identify new TBEV natural foci and genetically characterize strains in ticks in previously nonendemic areas in Eastern Germany and Western Poland. (2) Methods: Ticks were collected from vegetation in areas reported by TBE patients. After identification, ticks were tested for TBEV in pools of a maximum of 10 specimens using real-time RT-PCR. From the positive TBEV samples, E genes were sequenced. (3) Results: Among 8400 ticks from 19 sites, I. ricinus (n = 4784; 56.9%) was predominant, followed by D. reticulatus (n = 3506; 41.7%), Haemaphysalis concinna (n = 108; 1.3%), and I. frontalis (n = 2; <0.1%). TBEV was detected in 19 pools originating in six sites. The phylogenetic analyses revealed that TBEV strains from Germany and Poland clustered with other German strains, as well as those from Finland and Estonia. (4) Conclusions: Although there are still only a few cases are reported from these areas, people spending much time outdoors should consider TBE vaccination.

Interactions between hard ticks (Ixodidae) and bacterial tick-borne pathogens.

In Europe, ticks are particularly important vectors of pathogens known as tick-borne pathogens (TBP). TBP can influence hosts, including domestic animals and humans as well as ticks. This review focuses on interactions between hard ticks and medically and veterinary significant bacterial pathogens i.e. Borrelia burgdorferi s.l., Anaplasma spp, and Rickettsia spp. The interactions between ticks and bacteria include among others the impact on gene expression and tick behaviour. Infection with TBP may influence tick salivary proteins and midgut receptors. Infection with B. burgdorferi s.l. changes the bahaviour of the tick allowing them for longer questing and increased mobility, while A. phagocytophilum increases survive in low temperatures by upregulating the expression of antifreeze glycoprotein (IAFGP). Whereas Rickettsia spp. increases ticks attraction towards the 900 MHz electromagnetic field.

Dermacentor reticulatus - a tick on its way from glacial refugia to a panmictic Eurasian population.

The ornate dog tick (Dermacentor reticulatus) shows a recently expanding geographic distribution. Knowledge on its intraspecific variability, population structure, rate of genetic diversity and divergence, including its evolution and geographic distribution, is crucial to understand its dispersal capacity. All such information would help to evaluate the potential risk of future spread of associated pathogens of medical and veterinary concern. A set of 865 D. reticulatus ticks was collected from 65 localities across 21 countries, from Portugal in the west to Kazakhstan and southern Russia in the east. Cluster analyses of 16 microsatellite loci were combined with nuclear (ITS2, 18S) and mitochondrial (12S, 16S, COI) sequence data to uncover the ticks' population structures and geographical patterns. Approximate Bayesian computation was applied to model evolutionary relationships among the found clusters. Low variability and a weak phylogenetic signal showing an east-west cline were detected both for mitochondrial and nuclear sequence markers. Microsatellite analyses revealed three genetic clusters, where the eastern and western cluster gradient was supplemented by a third, northern cluster. Alternative scenarios could explain such a tripartite population structure by independent formation of clusters in separate refugia, limited gene flow connected with isolation by distance causing a "bipolar pattern", and the northern cluster deriving from admixture between the eastern and western populations. The best supported demographic scenario of this tick species indicates that the northern cluster derived from admixture between the eastern and western populations 441 (median) to 224 (mode) generations ago, suggesting a possible link with the end of the Little Ice Age in Europe.

Update on prevalence of Babesia canis and Rickettsia spp. in adult and juvenile Dermacentor reticulatus ticks in the area of Poland (2016-2018).

Ornate dog tick, Dermacentor reticulatus is an important vector of Babesia canis, and Rickettsia spp. and other pathogens of veterinary and public health interest. The current study is the first to investigate the long-term changes in prevalence of these pathogens in expanding tick populations in Central Europe. Molecular techniques (PCR, sequencing) were applied for the detection of pathogen DNA in adult (n = 2497) and juvenile ticks (1096 larvae and 410 nymphs). DNA of Rickettsia spp. was identified in 35% of adults and 12.6% of juvenile ticks. DNA of B. canis was detected in 3% of adult ticks and only in ticks from the Eastern region (regional prevalence 6%). As previously, no B. canis-positive ticks were found in Western Poland, including ticks from Wroclaw area (n = 298). DNA of B. canis was identified in 0.33% of juvenile ticks (in 3 pools of larvae and 2 nymphs) from the Eastern region. In the current study we confirmed high occurrence of R. raoultii in adults ticks from all four zones and relatively high prevalence of B. canis in the Eastern population of D. reticulatus, corresponding well with high incidence of canine babesiosis in this area of Poland. Finally, we confirmed R. raoultii and B. canis infection in all life stages of D. reticulatus ticks.

The influence of local environmental factors in southwestern Poland on the abundance of Ixodes ricinus and prevalence of infection with Borrelia burgdorferi s.l. and B. miyamotoi.

Ticks are important ectoparasites and vectors of pathogens that cause disease in humans and animals. The natural habitat of Ixodes ricinus ticks is forests, which are convenient habitats to search for hosts, including reservoir hosts, and therefore can be an important habitat source of tick-borne pathogens. The aim of the study was to assess the usefulness of detailed forest habitat-type maps to estimate the tick-borne risk at a local scale (Lower Silesia, SW Poland). For the purposes of estimating tick abundance, we used the land cover maps available from the Forest Data Bank. For I. ricinus collection, nine sites located in three forest habitat types were chosen: broadleaf forest, mixed broadleaf and coniferous forest and coniferous forest. Ticks were collected once a month from April to June 2018 and 2019 using the standard flagging method. At each of the nine sites, ticks were collected in four plots, of 100 m2 each. Tick abundance was analysed using general linear mixed models (GLMM). A total of 2196 (10.1/100 m2) ticks were collected, including 2093 Ixodes ricinus (95.3%; 9.6/100 m2), 46 Dermacentor reticulatus (2.1%; 0.2/100 m2) and 57 Haemaphysalis concinna (2.6%; 0.3/100 m2). Among the collected I. ricinus were 589 larvae (28.1%; 2.7/100 m2), 1261 nymphs (60.3%; 5.8/100 m2), 128 females (6.1%; 0.6/100 m2) and 115 males (5.5%; 0.5/100 m2). We found a highly significant effect of forest habitat type on the density of ticks for broadleaf forest (coefficient = 1.87267, p-value = 2.79e - 07). Additionally, a significant influence of air temperature and relative humidity on the abundance of ticks was observed. During spring, the peak activity of I. ricinus was recorded in May and June. For DNA amplification of Borrelia burgdorferi s.l., a nested PCR method was used. Out of 494 I. ricinus, 83 (16.8%) were positive for Borrelia spp. The RFLP method showed the occurrence of five species including four belonging to the B. burgdorferi s.l. complex: B. afzelii (30.1%), B. garinii (38.6%), B. valaisiana (2.4%) and B. lusitaniae (18.1%). Furthermore, B. miyamotoi (9.6%), a species belonging to bacteria that cause relapsing fever as well as co-infection of B. miyamotoi/B.lusitaniae (1.2%) were found. The differences in the infection level of Borrelia spp. between broadleaf forest and mixed broadleaf and coniferous forest were statistically significant.

Monitoring the expansion of Dermacentor reticulatus and occurrence of canine babesiosis in Poland in 2016-2018.

BACKGROUND: The significance of tick-borne diseases has increased considerably in recent years. Because of the unique distribution of the tick species Dermacentor reticulatus in Poland, comprising two expanding populations, Eastern and Western that are separated by a Dermacentor-free zone, it is important to conduct studies on the process of tick expansion and emergence of canine babesiosis. The main aim of the current study was to monitor the expansion of D. reticulatus populations from spring 2016 to autumn 2018 to determine (1) the actual geographical range of this tick species, and (2) and the seasonal/annual shift in range limits and changes in distance between Western and Eastern populations of ticks (the size of the non-endemic area). METHODS: Ticks were collected in spring/autumn during a 3-year study. From each season and year at least three pairs of sites from the Western and Eastern populations were selected. Then the mean distance between paired sites was calculated for each season and year. We collected and analyzed data from veterinary clinics on the number of canine babesiosis cases treated in the clinic during a whole year (2018). RESULTS: Accordingly, further expansion of the two D. reticulatus populations was recorded, mainly along river basins. Marked colonization of the gap zone was observed, with a mean annual shift in the range of 2.5-10 km and a steadily decreasing distance between the two tick populations. The occurrence of babesiosis in different regions revealed low numbers of cases in Western Poland (19 cases/year) and the gap area (only 7 cases/year) and high incidence (up to 250 cases/1000 dogs) and fatality (total 3.65%) in Central and Eastern Poland. Strong associations were found geographically between tick and babesiosis occurrence and temporally in the seasonal patterns of occurrence of ticks and outbreaks of babesiosis. CONCLUSIONS: We documented the shift in range limits and continued process of colonization of the gap zone accompanied by the emergence of canine babesiosis in the Eastern expansion zone. Updated maps of the distribution of ticks and occurrence of babesiosis in different regions of Poland have allowed us to predict of the emergence of pathogens vectored by D. reticulatus. Incidence (per 1000 dogs) of canine babesiosis in veterinary clinics by current range of D. reticulatus.

The local-scale expansion of Dermacentor reticulatus ticks in Lower Silesia, SW Poland.

The range of D. reticulatus is discontinuous in Europe, with a gap between the Western and Eastern European populations. Recent studies have shown, however, a decrease in the gap as a consequence of D. reticulatus spreading to new areas. This study aims to analyze the dynamic of local-scale changes in the D. reticulatus range in Lower Silesia, SW Poland. All sites of D. reticulatus presence recognized in our research were located in the north-western part of the study area (Wroclaw and its surroundings), whereas the south-eastern part was found to be free of these ticks. However, a five-year observation period (2014-2019) indicates the expansion of D. reticulatus on a local scale, with a general tendency to expand to the east, with northerly or southerly deviations from year to year. The settled sites differed in distance to the nearest built-up area, the density of resident population, as well as land development intensity in the immediate vicinity. The 100% probability isolines of D. reticulatus presence in Wroclaw and its surroundings allowed the determination of the rate of range change, which turned out to be uneven in terms of direction and speed. The average rate of change in the range of tick occurrence was estimated at 7 km in 3 years. A more accurate analysis of the estimated range changes-made using modeling and verification of predicted changes in the field-showed that the likely rate of range change can be estimated at around 0.6-2.3 km/year.

Sensitivity of Ixodes ricinus (L., 1758) and Dermacentor reticulatus (Fabr., 1794) ticks to entomopathogenic fungi isolates: preliminary study.

Entomopathogenic fungi of the genus Beauveria and Metarhizium play an important role in controlling the population of arthropods. However, the data on their effectiveness against ticks focus mainly on species that do not occur in Europe. The aim of the study was to assess the effectiveness of entomopathogenic fungi against two of the most important tick species in Europe: Ixodes ricinus and Dermacentor reticulatus. In our study, the majority of tested entomopathogenic fungi strains showed potential efficacy against both tick species; however, D. reticulatus was less susceptible in comparison to I. ricinus. The observed mortality of ticks was up to 100% by using all commercial strains as well as three out of nine of the environmental strains. Among all tested fungi, the most effective against both tick species was environmental strain Metarhizium anisopliae LO4(1) with LC50 values: 2.6 × 103 cfu/ml-5.7 × 105 cfu/ml. Botanigard proved to be more effective than MET52 with LC50 values: 6.8 × 103 cfu/ml-3.3 × 106 cfu/ml. The conducted bioassays indicate the potential possibility of using the environmental isolates of entomopathogenic fungi, as well as commercial strains in control of local populations of I. ricinus and D. reticulatus; however, the possibility of using them in vivo requires more research.

First record of Haemaphysalis concinna (Acari: Ixodidae) in Lower Silesia, SW Poland.

Haemaphysalis concinna Koch is one of 19 species of the genus Haemaphysalis which has been reported in the Palearctic region. In Europe, the presence of H. concinna ticks has been reported in numerous countries. In Poland, to date, the precise occurrence of H. concinna was known only from one site in the north-western region. This paper shows that H. concinna ticks can be considered a typical example of the tick fauna occurring in Lower Silesia, SW Poland. Tick monitoring was conducted using a standard flagging method in 24 sites in the various forest types of Lower Silesia. Among 1622 host-seeking ticks collected, H. concinna accounted for 2.7%. From the collected H. concinna there were: 25 (58.1%) larvae, 15 (34.9%) nymphs, 1 (2.3%) female, and 2 (4.7%) males. The presence of H. concinna was confirmed in 6 out of 24 tested sites in fresh mixed broadleaf forests, fresh mixed coniferous forests and in pastures.

Sensitivity of Ixodes ricinus (L., 1758) and Dermacentor reticulatus (Fabr., 1794) ticks to Bacillus thuringiensis isolates: preliminary study.

Bacillus thuringiensis is a highly specific entomopathogenic microorganism. Although defined as having properties which work against insects, its role in the control of tick populations is still insufficiently known. In our bioassay, four environmental strains of B. thuringiensis, along with one commercially available product (Vectobac), have been used against ticks. Vectobac turned out to be ineffective in the biocontrol of ticks; however, two of environmental B. thuringiensis strains proved to be efficient against both Ixodes ricinus and Dermacentor reticulatus. In those cases, the mortality rate for ticks was assessed as being up to 80%, and LC50 ranged between 9.1 × 106 and 1.3 × 1015 (cfu/ml). Dermacentor reticulatus males were the most sensitive to bacteria. The similarity between the most and least efficient B. thuringiensis strains in enzymatic profiles-including lipases, phosphatases, proteases, and chitinases-may indicate a limited role of detected enzymes in the pathogenicity profile of bacterial strains against ticks.

Seroprevalence of Borrelia burgdorferi in forest workers from inspectorates with different forest types in Lower Silesia, SW Poland: preliminary study.

To estimate the Lyme borreliosis (LB) risk for forest workers, totally 646 blood samples were tested for IgG and IgM anti-Borrelia burgdorferi s.l. (anti-B.b.) antibody occurrence using ELISA tests confirmed with western blot. To clarify the varied LB risk, additionally, the data from the Forest Data Bank determining the detailed forest habitat type in particular forest inspectorates were used. The occurrence of the anti-B.b. antibody was confirmed in 22% (8.7% IgM, 17.8% IgG) of forest workers. Analysis of the influence of the habitat type (forest types) indicated the significant positive impact of the occurrence of the deciduous and mixed-deciduous forests on the seroprevalence of anti-B.b. IgG level among forestry workers. However, the share of forest type cannot be the only factor taken into account when assessing risk.

Local-scale spatio-temporal distribution of questing Ixodes ricinus L. (Acari: Ixodidae)-A case study from a riparian urban forest in Wroclaw, SW Poland.

This paper presents the distribution of questing Ixodes ricinus ticks in suburban forest intensively visited by people. The local-scale observations conducted during a 4-year study at 99 plots (of 100m2 each) located throughout the entire area of a riparian urban forest, showed a high variation in the density of ticks from year to year. Although I. ricinus is generally permanent in the study area, spatial distribution of sample plots harbouring I. ricinus is variable, i.e. mainly random for adults and larvae, and random or clustered for nymphs. Among the most common plant species in the herb layer, there were not any species which had a statistically significant and constant impact on the occurrence of any of the development stages of I. ricinus. Also relations between the density of tick development stages and vegetation variables, including cover of the herb layer, total species number, species number of the herb layer, and percentage coverage of particular species, as well as ecological indices for light, soil moisture, reaction, and nutrients, did not show any constant and predictable pattern in subsequent years of the study. Only tree and shrub layers were found as variables positively affecting the density of ticks. Although small, suburban forests can be considered as tick-borne risk areas, it is impossible to determine in details areas of tick-borne risk.

Winter activity of Dermacentor reticulatus (Fabricius, 1794) in the newly emerging population of Lower Silesia, south-west Poland.

This paper presents the unexpected winter activity of Dermacentor reticulatus (Fabricius, 1794) ticks in a newly emerging population in south-west Poland. Host-seeking ticks were collected from vegetation in January 2016 in a meadow ecosystem in six sites located in the Wroclaw Agglomeration, as well as from ten companion animals. A total of 238 questing D. reticulatus ticks, comprising 166 females and 72 males, were collected from all examined sites with the highest number of 102 specimens collected in one hour in one locality (Muchobór Wielki, Wroclaw). Additionally, two fully-engorged females were collected from two dogs along with one slightly engorged female from a cat. The fact that D. reticulatus can be very active in January indicates a need to take into account the increased threat of tick-bite in the winter time.

Students' attitudes to tick risks.

INTRODUCTION AND OBJECTIVES: The ever-increasing number of patients with tick-borne diseases resulted in the presented study investigating the awareness, attitudes and knowledge among students about the threats arising from tick bites and preventive anti-tick practices. MATERIALS AND METHOD: Questionnaires concerning these issues were distributed amongst Czech and Polish university students of science. Responses were analyzed by nationality and by gender. RESULTS: Nearly all respondents were aware of the risks arising from ticks and could name at least one disease transmitted by ticks. The Czech students felt more threatened by tick-borne diseases, had more frequently suffered from Lyme borreliosis and were vaccinated against tick-borne encephalitis more often than the Polish students. A large number of the participants applied preventive measures against ticks in order to protect themselves. The Czech students believed in the effectiveness of repellents statistically more often than the Polish students, while effectiveness is the main criterion for selection of the right repellent in both groups. CONCLUSION: Differences in preferences between the two nations appeared in many areas, e.g. the Czechs felt more threatened by all kind of risks and suffered from Lyme disease more frequently. Gaps can still be found in both the knowledge and behaviour among the respondents. It can be expected that the general public knowledge of this issue is rather limited in comparison with the students participating in the study, who are systematically educated in the field.

Dermacentor reticulatus (Fabricius, 1794) and Babesia canis (Piana et Galli-Valerio, 1895) as the parasites of companion animals (dogs and cats) in the Wroclaw area, south-western Poland.

UNLABELLED: Tests performed in 2013 and 2014 revealed the occurrence of three tick species parasitizing pet cats and dogs in the Wroclaw Agglomeration. In total, 1,455 tick specimens were removed from 931 hosts (760 dogs and 171 cats) in 18 veterinary clinics. The dominant tick species was Ixodes ricinus (n=1272; 87.4%), followed by I. hexagonus (n=137; 9.4%) and Dermacentor reticulatus (n=46; 3.2%). Females were the most often collected development stage among I. ricinus and D. reticulatus, and nymphs among I. hexagonus. Additionally, D. reticulatus ticks (n=337) were then collected from vegetation in the Wroclaw area to detect Babesia canis; however, none was found positive. Only 9.0% of dog blood samples sent to VETLAB were positive for Babesia spp. Negative results for B. canis from ticks may result from the short period of the occurrence of D. reticulatus in the Wroclaw area and therefore the vectorpathogen cycle may not have been fully established at the time of the study. Nevertheless, D. reticulatus is expanding its range, and the size of its population in the Wroclaw Agglomeration is increasing. The presence of the pathogenic Babesia spp. combined with the occurrence of its main vector¸ D. reticulatus, suggests that the epizootiological situation in the area can change and may pose a new veterinary problem in the future. KEY WORDS: Dermacentor reticulatus, Babesia canis, pets, Wroclaw, Poland.