Parasitic Arthropods of Soricinae Shrews in North-Eastern Poland.

The study of the ectoparasite fauna of the insectivores-Sorex araneus, Sorex minutus, Neomys fodiens, and Neomys anomalus (subfamily Soricinae)-was carried out in three locations in Poland: Bialowieza National Park, Kosewo Górne in the Masurian Lake District, and in vicinity of Warsaw. Three species of Ixodidae ticks, eleven species of fleas, and four species of mites from the order Mesostigmata were noted. The most numerous ectoparasites are ticks Ixodes ricinus (larvae), Dermacentor reticulatus (nymphs), and fleas Palaeopsylla soricis, Megabothris walkeri, and Hystrichopsylla orientalis. These species show the highest prevalence and show the highest dominance index. The parasitofauna of S. araneus is much richer in species than other shrew species. The structure and dominance of parasite assemblages differ between locations.

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.

Molecular identification of Trypanosoma theileri complex in Eurasian moose Alces alces (L.).

Although the significance of red deer (Cervus elaphus) and roe deer (Capreolus capreolus) as hosts and their role in the circulation of vector-borne pathogens in Europe is well described, the trypanosomes of moose (Alces alces) are poorly known. As heat sensitive ungulates, moose might be especially vulnerable to the effects of climate change and the associated rise in parasite pressure. Therefore, the aim of our study was to determine the prevalence of trypanosomes in moose in Poland, this being one of the largest populations in Central Europe since the 2001 hunting ban. Molecular analysis revealed the presence of Megatrypanum trypanosomes in almost half of the studied moose. As the population of moose in Central Europe has been recently growing, it is crucial to determine their role in the circulation of vector-borne pathogens in environment. This is the first study of the detection and molecular identification of Trypanosoma theileri complex in moose in central Europe.

The role of sheep ked (Melophagus ovinus) as potential vector of protozoa and bacterial pathogens.

The sheep ked (Melophagus ovinus) hematophagous insect may act as a potential vector of vector-borne pathogens. The aim of this study was to detect the presence of Trypanosoma spp., Bartonella spp., Anaplasma phagocytophilum and Borrelia burgdorferi sensu lato in sheep ked collected from sheep in Poland. In total, Trypanosoma spp. was detected in 58.91% of M. ovinus, whereas Bartonella spp. and B. burgdorferi s.l. were found in 86.82% and 1.55% of the studied insects, respectively. A. phagocytophilum was not detected in the studied material. In turn, co-infection by Trypanosoma spp. and Bartonella spp. was detected in 50.39%, while co-infection with Trypanosoma spp. and Bartonella spp. and B. burgdorferi s.l. was found in 1.55% of the studied insects. The conducted study showed for the first time the presence of B. burgdorferi s. l. in M. ovinus, as well as for the first time in Poland the presence of Trypanosoma spp. and Bartonella spp. The obtained results suggest that these insects may be a potential vector for these pathogens, but further-more detailed studies are required.

Molecular Detection of Bartonella spp. in Rodents in Chernobyl Exclusion Zone, Ukraine.

PURPOSE: Bacteria of the genus Bartonella are obligate parasites of vertebrates. Their distribution range covers almost the entire world, from the Americas to Europe and Asia. Many Bartonella species use rodents as reservoirs, and while much is known about Bartonella infection of rodents in central Europe, its extent is poorly understood in Eastern Europe. METHODS: The present study examines five rodent species (Apodemus flavicollis, Myodes glareolus, Microtus arvalis, Apodemus agrarius, Apodemus sylvaticus) in the Chernobyl Exclusion Zone in Ukraine. Total of 36 small mammals were captured in September 2017. RESULTS: The overall prevalence of Bartonella spp. was 38.9% (14/36) in rodents. Obtained four sequences from Apodemus flavicollis, were identical to Bartonella grahamii and B. taylorii. CONCLUSION: This is the first report to confirm the presence of Bartonella spp. in rodents in the Chernobyl Exclusion Zone, Ukraine by molecular methods. The sequences show similarity to Bartonella strains occurring in Europe.

Molecular detection of Trypanosoma spp. in Lipoptena cervi and Lipoptena fortisetosa (Diptera: Hippoboscidae) and their potential role in the transmission of pathogens.

The family Hippoboscidae is a less known group of blood-sucking flies. Deer ked are particularly important for animal health; they may act as potential vectors of disease to ungulates, and may transmit pathogens to animals and humans. The aim of this study was to investigate the presence of Trypanosoma (Megatrypanum) DNA in deer keds using molecular methods. Results prove the presence of Megatrypanum trypanosome DNA in the studied winged adult deer keds and this is the first detection of this pathogen in Lipoptena fortisetosa. In addition, this paper evidences the occurrence of L. fortisetosa in two new locations: one in the Bialowieza Primeval Forest, and another in the Strzalowo Forest Inspectorate (Piska Forest), both in north-eastern Poland.

Ticks (Acari: Ixodidae) Parasitizing Red Foxes (Vulpes vulpes) in Slovakia and New Data About Subgenus Pholeoixodes Occurrence.

BACKGROUND: Distribution and biology of Pholeoixodes ticks is not very well understood. The goal of the study was to collect new data on the Pholeoixodes tick occurrence in Slovakia. METHODS: Tick infestation of red foxes in the regions of Kosice, Presov, Bratislava and Zilina was studied during the period 2017-2018. Ticks were collected from the fur of animals using tweezers and identified using appropriate keys. In total, 146 red foxes (Vulpes vulpes) were investigated. RESULTS: In total, 39 (26.7%) of animals were found to be infected with ticks from five species. Pholeoixodes ticks were found on 13 (3.4%) of the foxes: Ixodes hexagonus (Leach, 1815) on 5 specimens (3.4%), in the Kosice, Presov and Zilina regions; I. crenulatus (Koch, 1844) on 8 specimens (5.5%) in the Presov and Bratislava regions; Ixodes ricinus (Linnaeus, 1758) collected from 25 (17.2%) foxes in every locality; Dermacentor reticulatus (Fabricius, 1794) from 5 foxes (3.4%) in the Kosice, Presov and Zilina regions; Haemaphysalis concinna (Koch, 1844), from 4 foxes (2.8%) from the Kosice region. CONCLUSIONS: Ixodes hexagonus has been previously recorded in Slovakia. However, this is the first finding of I. crenulatus in the country. The morphological features of the I. crenulatus specimens found in Slovakia were identical to those of ticks described in Poland and descriptions given in identification keys.

Molecular detection of Anaplasma phagocytophilum in wild carnivores in north-eastern Poland.

BACKGROUND: Anaplasma phagocytophilum is an obligate parasitic intracellular bacterium. It is the causative agent of granulocytic anaplasmosis, with effects on human and animal health. In Europe, the pathogen is mainly transmitted among a wide range of vertebrate hosts by blood-sucking arthropods. The aim of this study was to determine the presence of A. phagocytophilum in wild carnivores, viz raccoon dogs (Nyctereutes procyonoides), badgers (Meles meles), foxes (Vulpes vulpes), martens (Martes sp.) and European polecats (Mustela putorius), using molecular methods. METHODS: In the present study, 174 spleen samples were collected from adult, wild carnivores hunted in the years 2013-2016. A short fragment (383 bp) of the 16S ribosomal RNA gene partial sequence was used as a marker to identify A. phagocytophilum in spleen samples collected from carnivores using nested PCR. RESULTS: The prevalence of A. phagocytophilum in wild carnivores was 31.61% (55/174). Seven sequences of A. phagocytophilum were generated from two raccoon dogs, two badgers, one marten, one red fox and one European polecat. Six identical nucleotide sequences were obtained from one raccoon dog, two badgers, one marten, one red fox and one European polecat (A. phagocytophilum sequences 1: MH328205-MH328209, MH328211), and these were identical to many A. phagocytophilum sequences in the GenBank database (100% similarity). The second sequence (A. phagocytophilum sequence 2: MH328210) obtained from the raccoon dog shared 99.74% identity with A. phagocytophilum sequence 1. CONCLUSIONS: To our knowledge, this is the first study to use molecular methods to determine the presence of A. phagocytophilum in wild carnivores, viz raccoon dog, badger, marten and European polecat, in Poland. The detected A. phagocytophilum sequences (1 and 2) were closely related with those of A. phagocytophilum occurring in a wide range of wild and domestic animals and vectors.

Molecular Detection of Anaplasma phagocytophilum in Blood-Sucking Flies (Diptera: Tabanidae) in Poland.

Anaplasma phagocytophilum is a pathogen of veterinary and medical importance. It is the causative agent of tick-borne fever (TBF) in ruminants (also known as bovine or ovine granulocytic anaplasmosis), and of human granulocytic anaplasmosis (HGA) in humans. In Europe, A. phagocytophilum is transmitted by Ixodes ricinus (Linnaeus 1758) ticks. The aim of this study was to confirm the presence of A. phagocytophilum DNA in blood-sucking flies belonging to the Tabanidae family using molecular methods. It represents the first detection of this pathogen in Haematopota pluvialis (Linnaeus 1758), Tabanus bromius (Linnaeus 1758), and Tabanus distinguendus (Verrall 1909) in Europe.

Rickettsiales Occurrence and Co-occurrence in Ixodes ricinus Ticks in Natural and Urban Areas.

Bacteria of Rickettsiaceae and Anaplasmataceae families include disease agents spread by Ixodes ricinus ticks, the most common tick vector in Europe. The aim of the study was to compare the prevalence and co-infection prevalence of particular tick-transmitted Rickettsiales members: Rickettsia spp. (further referred as Rs), Anaplasma phagocytophilum (Ap), and "Candidatus Neoehrlichia mikurensis" (CNM) in I. ricinus ticks in two types of areas, different in terms of human impact: natural and urban. Using additional data, we aimed at investigating co-occurrence of these Rickettsiales with Borreliella spp. A total of 4189 tick specimens, 2363 from the urban area (Warsaw park and forests) and 1826 from the natural area (forests and park in the vicinity of National Parks), were tested for the presence of Rickettsiales DNA by PCRs. The prevalence of selected Rickettsiales was twice higher in urban than natural areas (13.2% vs. 6.9%, respectively). In total ticks, the prevalence of Rs, Ap, and CNM was 6.5%, 5.3%, and 3.6% in urban areas vs. 4.4%, 1.1%, and 2.1% in natural areas, respectively. Co-infections of Rickettsiales were also more prevalent in urban areas (2.6% vs. 0.3%, respectively). The most common Rs was R. helvetica; also R. monacensis and novel "Candidatus Rickettsia mendelii" were detected. Positive association between Ap and CNM infections was discovered. Rickettsiales bacteria occurrence was not associated with Borreliella occurrence, but co-infections with these two groups were more common in ticks in urban areas. In conclusion, three groups of Rickettsiales constituted the important part of the tick pathogen community in Poland, especially in the urbanized central Poland (Mazovia). In the Warsaw agglomeration, there is a greater risk of encountering the I. ricinus tick infected with Rickettsiales and co-infected with Lyme spirochaetes, in comparison to natural areas. This finding raises the question whether cities might in fact be the hot spots for TBDs.

The Competition Between Immatures of Ixodes ricinus and Dermacentor reticulatus (Ixodida: Ixodidae) Ticks for Rodent Hosts.

The numbers of Ixodes ricinus (L.) and Dermacentor reticulatus (Fabricius) larvae and nymphs attached to small mammals are influenced by interspecific competition. The present study analyses data collected over several years in two study areas: Kosewo Górne (Mazurian District, N Poland; between July 1997 and July 2009) and Bialowieza Primeval Forest (E Poland; in July 2007). In total, 975 ticks were collected from striped field mice (Apodemus agrarius), yellow-necked mice (A. flavicollis), bank voles (Myodes glareolus), and root voles (Microtus oeconomus). In total, of the 203 investigated rodents, 137 were infested with ticks and 39 demonstrated mixed infection. The numbers of the two tick species found on Apodemus mice were significantly negatively correlated with those on root voles; similarly, although bank voles were significantly more frequently infested by I. ricinus than by D. reticulatus, the reverse was observed in root voles. In addition, among the voles, each tick species was found in different locations on the host body, which could also result from competitive interactions; furthermore, competitive release regarding microhabitat selection was observed on hosts infested with one tick species. This competition may be driven by the limited area of host body available for foraging by ticks, i.e., safe areas of high vascularization covered by thin skin. However, the mechanisms of such competition require further investigation.

The role of particular ticks developmental stages in the circulation of tick-borne pathogens in Central Europe. 5. Borreliaceae

The developmental cycles of all B. burgdorferi s.l. genospecies present typical, main pattern described in the 90thies. The simple scheme might be modified according to the biology of species and hosts preference. Central European genospecies of B. burgdorferi s.l. can be associated with four groups of hosts playing the role of animal reservoirs. The group 1 contains genospecies associated with rodents as primary animal reservoir ­ B. afzelii, B. garinii, B. burgdorferi sensu stricto, strains B. bavariensis (B. garinii OspA serotype 4). The group 2 involves B. valaisiana and most of B. garinii strains, associated with birds. The group 3 involves B. spielmanii, the reservoir hosts are Gliridae, and hedgehogs. The group 4 includes B. lusitaniae, the hosts are lizards. B. miyamotoi enzootic cycle seems to be similar to B. burgdorferi complex, however, differs by the transovarial transmission possibility. The divisions are not extreme; in the hosts group, infected with appropriate Borrelia genospecies, very often are found the specimens infected with other genospecies.

Microsatellite-based genetic diversity of Dermacentor reticulatus in Europe.

Dermacentor reticulatus, also known as ornate dog tick, is an important vector of the causative agents of various tick-borne diseases in humans, livestock and wild animals. The geographical range of D. reticulatus in Europe is fragmented and divided into two main zones: The Western European and the Eastern European zone. To investigate D. reticulatus population structure through its distribution range we used microsatellite markers and compared the genetic diversity of D. reticulatus in 26 selected sites in the western and eastern European distribution areas. A total of 254 unfed adult D. reticulatus ticks were collected from vegetation and genotyped at 5 microsatellite loci, and altogether 26 alleles were detected. The overall FST value of pairwise comparisons among 26 sampling sites was 0.128 and revealed a moderate genetic differentiation. Bayesian-clustering analysis showed that D. reticulatus forms two genetically distinct groups across Europe. Division of D. reticulatus distribution range into Eastern European and Western European populations with a possible recent overlap in Poland has been supported by molecular data obtained in this study.

The role of particular ticks developmental stages in the circulation of tick-borne pathogens in Central Europe. 6. Babesia.

In the Central European conditions, three species of Babesia have epidemiological significance as human pathogens ­ Babesia divergens, B. microti and B. venatorum. Tick Ixodes ricinus is considered as their main vector, wild mammals as the animal reservoir. The zoonotic cycles of small and large Babesia differ in details. Due to the lack of transovarial mode transmission in small species B. microti, the circulation goes mainly between immature ticks and vertebrate hosts; pathogen circulates primarily in the cycle: infected rodent → the tick larva → the nymph → the mammal reservoir →the larva of the tick. The tick stages able to effectively infect human are nymphs and adult females, males do not participate in the follow transmission. For large Babesia ­ B. divergens and B. venatorum, the transovarial and transstadial transmission enable the presence of the agent in adult ticks, moreover, that larvae and nymphs feed on not-susceptible hosts. The tick stages able to effectively infect cattle and other ruminants are adult females. Resuming, pathogen circulates primarily in the cycle the ruminant host ­ adult female tick ­ the larva ­ the nymph ­ adult female of the next generation ­ the ruminant. Due to the compound developmental transmission has place after the outflow of a tick began feeding.

Ticks and the city - are there any differences between city parks and natural forests in terms of tick abundance and prevalence of spirochaetes?

BACKGROUND: Ixodes ricinus ticks are commonly encountered in either natural or urban areas, contributing to Lyme disease agents Borreliella [(Borrelia burgdorferi (sensu lato)] spp. and Borrelia miyamotoi enzootic cycles in cities. It is an actual problem whether urbanization affects pathogen circulation and therefore risk of infection. The aim of the study was to evaluate main tick-borne disease risk factors in natural, endemic areas of north-east (NE) Poland (Bialowieza) and urban areas of central Poland (Warsaw), measuring tick abundance/density, prevalence of infection with spirochaetes and diversity of these pathogens in spring-early summer and late summer-autumn periods between 2012 and 2015. METHODS: Questing I. ricinus ticks were collected from three urban sites in Warsaw, central Poland and three natural sites in Bialowieza, NE Poland. A total of 2993 ticks were analyzed for the presence of Borreliella spp. and/or Borrelia miyamotoi DNA by PCR. Tick abundance was analyzed by General Linear Models (GLM). Prevalence and distribution of spirochaetes was analyzed by Maximum Likelihood techniques based on log-linear analysis of contingency tables (HILOGLINEAR). Species typing and molecular phylogenetic analysis based on the sequenced flaB marker were carried out. RESULTS: Overall 4617 I. ricinus ticks were collected (2258 nymphs and 2359 adults). We report well established population of ticks in urban areas (10.1 ± 0.9 ticks/100 m2), as in endemic natural areas with higher mean tick abundance (16.5 ± 1.5 ticks/100 m2). Tick densities were the highest in spring-early summer in both types of areas. We observed no effect of the type of area on Borreliella spp. and B. miyamotoi presence in ticks, resulting in similar prevalence of spirochaetes in urban and natural areas [10.9% (95% CI: 9.7-12.2%) vs 12.4% (95% CI: 10.1-15.1%), respectively]. Prevalence of spirochaetes was significantly higher in the summer-autumn period than in the spring-early summer [15.0% (95% CI: 12.8-17.5%) vs 10.4% (95% CI: 9.2-11.6%), respectively]. We have detected six species of bacteria present in both types of areas, with different frequencies: dominance of B. afzelii (69.3%) in urban and B. garinii (48.1%) in natural areas. Although we observed higher tick densities in forests than in maintained parks, the prevalence of spirochaetes was significantly higher in the latter [9.8% (95% CI: 8.6-11.0%) vs 17.5% (95% CI: 14.4-20.5%)]. CONCLUSIONS: Surprisingly, a similar risk of infection with Borreliella spp. and/or B. miyamotoi was discovered in highly- and low-transformed areas. We suggest that the awareness of presence of these disease agents in cities should be raised.

Molecular detection of Bartonella spp. in deer ked (Lipoptena cervi) in Poland.

BACKGROUND: The bacteria of the genus Bartonella are obligate parasites of vertebrates. Their distribution range covers almost the entire world from America, Europe, Asia to Africa and Australia. Some species of Bartonella are pathogenic for humans. Their main vectors are blood-sucking arthropods such as fleas, ticks and blood-feeding flies. One such dipteran able to transfer vector-borne pathogens is the deer ked (Lipoptena cervi) of the family Hippoboscidae. This species acts as a transmitter of Bartonella spp. in cervid hosts in Europe. METHODS: In the present study, 217 specimens of deer ked (Lipoptena cervi) were collected from 26 red deer (Cervus elaphus) hunted in January 2014. A short fragment (333 bp) of the rpoB gene was used as a marker to identify Bartonella spp. in deer ked tissue by PCR test. A longer fragment (850 bp) of the rpoB gene was amplified from 21 of the positive samples, sequenced and used for phylogenetic analysis. RESULTS: The overall prevalence of Lipoptena cervi infection with Bartonella spp. was 75.12% (163/217); 86.67% (104/120) of females and 60.82% (59/97) of males collected from red deer hunted in the Strzalowo Forest District in Poland (53°45'57.03″N, 21°25'17.79″E) were infected. The nucleotide sequences from 14 isolates (Bartonella sp. 1) showed close similarity to Bartonella schoenbuchensis isolated from moose blood from Sweden (GenBank: KB915628) and human blood from France (GenBank: HG977196); Bartonella sp. 2 (5 isolates) and Bartonella sp. 3 (one isolate) were similar to Bartonella sp. from Japanese sika deer (GenBank: AB703149), and Bartonella sp. 4 (one isolate) was almost identical to Bartonella sp. isolated from Japanese sika deer from Japan (GenBank: AB703146). CONCLUSIONS: To the best of our knowledge, this is the first report to confirm the presence of Bartonella spp. in deer keds (Lipoptena cervi) in Poland by molecular methods. Bartonella sp. 1 isolates were most closely related to B. schoenbuchensis isolated from moose from Sweden and human blood from France. The rest of our isolates (Bartonella spp. 2-4) were similar to Bartonella spp. isolated from Japanese sika deer from Japan.

Origins of recently emerged foci of the tick Dermacentor reticulatus in central Europe inferred from molecular markers.

The ornate dog tick Dermacentor reticulatus is vector of several blood parasites, including Babesia canis, a causative agent of babesiosis. The geographical range of D. reticulatus in Europe is discontinuous with a gap separating eastern and western macroregions. New foci observed in several locations in western and central Europe were considered an expansion of the western population, including foci in western Poland. In the present paper we used molecular markers to identify the origins of these foci, and we compared their genetic polymorphism to D. reticulatus collected in sites situated within the eastern population. The overall polymorphism in mt 16S rDNA was low, and all sites from the western population shared the same haplotype suggesting the expansion in this area. In the marker 5.8S rDNA-ITS2 we found no differences in polymorphism between sites from eastern Poland (eastern population), and newly emerged foci in western Poland considered a putative expansion zone of the western population. However, the sites from western Poland differed considerably from nearby German site. Our results show that foci in western Poland could not have originated from D. reticulatus from the western population, as previously thought. We found that the state border following river hinders considerably gene flow between adjacent sites what suggest that natural dispersal of D. reticulatus by wildlife is unlikely, and the emergence of new foci should rather be contributed to human-associated dispersal. We propose that livestock, and pets travelling with their owners are the most probable source of new foci, and they can easily transfer ticks within a country but not between countries.

The role of particular tick developmental stages in the circulation of tick-borne pathogens affecting humans in Central Europe. 3. Rickettsiae.

UNLABELLED: Ixodes ricinus, Dermacentor reticulatus and D. marginatus ticks are the most important vector for Rickettsia spp. in Central Europe. Ticks sustain rickettsial transmission cycles transovarially and transstadially, it makes enable the rickettsial circulation in the tick population in the absence of vertebrate competent reservoir. Rickettsia helvetica is transmitted by I. ricinus tick; the highest rates of infection are noted in adult females, lower in males and in nymphs. All tick developmental stages apart males are able to infect mammal hosts and humans. The potential animal reservoir could be wild boar, the role of deer is unclear; small rodents maintain the tick population. Rickettsia slovaca is transmitted by D. marginatus and D. reticulatus ticks. The available data suggest the role of wild boars and Apodemus mice as animal reservoir. The ticks able to infect human are adults D. marginatus. Rickettsia raoultii is transmitted by D. marginatus and D. reticulatus. The infections of mammals are not recorded. As in Rickettsia slovaca, human can be infected by adults D. marginatus. Rickettsia monacensis is transmitted in Central Europe by I. ricinus tick (apart males), although there is a documented infection of Dermacentor ticks. The differences in the infection rates of tick's larvae, nymphs and adults suggest the limited role of transovarial transmission, and the participation of mammals in the zoonotic cycle, being the source of infection for larvae and nymphs. KEY WORDS: SFG, Rickettsia slovaca, Rickettsia helvetica, Rickettsia raoultii, Rickettsia monacensis, ticks.