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.

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.

Dominance of Dermacentor reticulatus over Ixodes ricinus (Ixodidae) on livestock, companion animals and wild ruminants in eastern and central Poland.

The most common tick species parasitizing animals in Poland are Ixodes ricinus and Dermacentor reticulatus. These tick species differ in their distribution, habitats, seasonal activity and host specificity. Ixodes ricinus is the most prevalent and widely distributed, whereas the range of D. reticulatus is limited to eastern and central parts of the country with several new foci in the middle-west and the west. However, as in many central European countries, the range of D. reticulatus is expanding, and some authors have correlated this expansion with an increasing number of available hosts. The aim of the present study was to determine the tick fauna on domestic and livestock animals in two areas endemic for I. ricinus and D. reticulatus and to compare the risk of infestation with different tick species in open and forest areas. Over a 14 month period, 732 ticks were collected from five host species including domestic animals (dogs and cats), livestock (cows and horses) and wildlife (European bison) in two areas, central and NE Poland, endemic for D. reticulatus. Three tick species were recorded: D. reticulatus (623 individuals; 85.1% of all collected ticks), I. ricinus (106 individuals; 14.5%) and three females of Ixodes hexagonus (0.4%) from a dog. Dermacentor reticulatus was the dominant tick species found on four host species and constituted 86, 81, 97 and 100% of all ticks from dogs, horses, cows and bison, respectively, and was collected from animals throughout the year, including during the winter. The common tick, I. ricinus, was the dominant tick collected from cats (94%). Fully-engorged, ready-for-reproduction females of D. reticulatus were collected from all host species. In May 2012, questing ticks were collected by dragging in forest or open habitats. The density of adult marsh ticks in open areas was around 2 ticks/100 m(2) in the majority of locations, with a maximum of 9.5 ticks/100 m(2). The density of adult I. ricinus was much lower in its typical habitat (forests: range 0.8-2.2 ticks/100 m(2)) between three and seven times lower than the density of D. reticulatus in its typical habitat. In regions endemic for marsh ticks, this tick species constitutes the main risk of tick infestation for livestock and dogs throughout the year. Livestock and companion animals are competent hosts for D. reticulatus, enabling the completion of the tick's life cycle. Anti-tick treatment should be adjusted to marsh tick seasonal activity and drug sensitivity.

Prevalence of tick-borne encephalitis virus (TBEV) RNA in Dermacentor reticulatus ticks from natural and urban environment, Poland.

Tick-borne encephalitis virus (TBEV) (Flaviviridae, Flavivirus) is an arthropod-borne virus, an etiologic agent of tick-borne encephalitis (TBE), a human infection involving the central nervous system. The disease is endemic in a large region in Eurasia, where it is transmitted mainly by Ixodes ricinus and Ixodes persulcatus ticks. It is known that also Dermacentor reticulatus is involved in circulation of TBEV. However, the current knowledge of D. reticulatus importance in TBE epidemiology is still insufficient. A total of 471 adult D. reticulatus ticks were collected by flagging vegetation in the Bialowieza Primeval Forest, Biebrza National Park, Masurian Landscape Park (North-Eastern Poland) and in the city of Warsaw in the years 2007-2010. All collected ticks were examined individually for the presence of RNA of TBEV using nested RT-PCR assay. Positive results were noted in all investigated localities with the infection rate ranging from 0.99 to 12.5 % with a total mean of 2.12 %. The difference in the percentage of infective males and females was not statistically significant.

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.

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.

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.

New locations of Dermacentor reticulatus ticks in Western Poland: the first evidence of the merge in D. reticulatus occurrence areas?

The ornate dog tick, D. reticulatus, is the second most common tick species in Europe. It occurs across the temperate zone of Eurasia, from England and France to the basin of the Jenisej River in Siberia, and the occurrence area ranges from 56-57 degrees N latitude down to 52 degrees N latitude. The range is divided into two distinct parts, the West-European and the Eastern part. The western region covers France and northern Spain, South-western England and Wales, Germany, Austria, Czech, Western Slovakia and Hungary. The eastern region extends from the eastern part of Poland and Slovakia, through Belarus and Russia as far east as Siberia. The southern parts of this region cover Ukraine, Eastern Hungary and Romania. From the 1970s, D. reticulatus has expanded its area of occurrence. In Poland, the front of the area is historically situated along the Vistula River. From the 1980s new tick populations has appeared to the west of this border. In the past years, new populations of D. reticulatus appeared close to the western border of Poland near Szczecin and Wroclaw. These are possibly the first observations of the merge between the two separated areas of D. reticulatus occurrence.

Blood parasites of mound-building mouse, Mus spicilegus Petényi, 1882 (Mammalia, Rodentia).

Mound-building mice, Mus spicilegus, were studied for the blood parasites in Eastern Slovakia, vicinity Kechnec village near Kosice town (Kosická kotlina basin, 21 degrees 14' E, 48 degrees 33' N) during years 2002-2005. Overall, 251 specimens were examined. The parasites were detected using microhematokrit centrifugation technique and on the Giemsa's method stained blood smears and light microscopy. The parasites were found in 3.57% of specimens; 1.20% of mice were infected with Bartonella sp., 2.39% were infected with Babesia piroplasms. No Hepatozoon hemogregarines and trypanosomes were observed. The intensity of infection with Bartonella was low, less than 0.01% of erythrocytes were invaded, the percent of the erythrocytes with Babesia sp. was less than 0.01%. The morphological description and measurements of parasites were made using the "Analysis" software combined with a video camera and a microscope. The mean size of Bartonella sp. bacteria's were 0.8 x 0.3 microm, range 0.4-1.5 x 0.1-0.9 microm, Babesia sp. occurred in pear-shaped and ring-like forms, 1.00-1.27 microm in diameter, and 0.98-1.27 microm in size, respectively. The regular form of four cells--"maltese cross" was not noticed. This is the first record infection of Mus spicilegus with blood parasites.

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.

Rickettsia spp. in ticks, Poland.

Ticks are recognized as the main vectors and reservoirs of spotted fever group rickettsiae. We searched for the most prevalent Rickettsia spp. in Poland and found R. slovaca and R. helvetica bacteria in ticks in southern and central Poland; R. raoulti was found in ticks in all parts of Poland.

The estimation scale of endangerment with tick attacks on recreational towns areas.

The 5-classed scale of threats with tick's attacks was modelled on the ground diversification of the number of tick collected. The areas above 50 ticks collected per hour, was named as the very high class, minimal level of endangerment (1 to 3 ticks) named low class, medium values were established: medium low class of endangerment, medium class of endangerment, high class of endangerment respectively.

The occurrence and ultrastructure of Trypanosoma (Herpetosoma) lewisi (Kent, 1880) Laveran and Mesnil, 1901, the parasite of rats (Rattus norvegicus) in Poland.

This study reports the light and electron microscopic examination of Trypanosoma (Herpetosoma) lewisi (Kent, 1880) Laveran and Mesnil, 1901, isolated from rats (Rattus norvegicus) from Poland. Bloodstream trypomastigotes were identified morphometrically from 100 specimens collected from three naturally infected rats Rattus norvegicus. Body length ranged from 15.45-23.64 microm and width from 1.3-2.32 microm while the free flagellum was 8.1 microm long. Electron microscopic study of bloodstream trypomastigotes exhibited typical ultrastructural features similar to those of other stercorarian trypanosomes. The presently determined morphological data have been compared with those provided by other authors.

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.

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.

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 case of babesiosis in farmed wolf (Canis lupus L).

There was recognised the case of canine babesiosis in farmed wolf (Canis lupus) in Bialowieza, northeastern Poland. The typical symptoms were observed in animal--depression, asitia, and haematocyturia. The treatment with antibiotics gave positive results. It was the first observation of babesiosis in wolf in Poland.