First molecular identification of Haemonchus contortus (Nematoda: Trichostrongylidae), a blood-sucking gastric nematode of artiodactyles, in the ground beetle Carabus granulatus (Coleoptera: Carabidae).

Among gastrointestinal nematodes, Haemonchus contortus (Rudolphi) Cobb (order Strongylidae; family Trichostrongylidae) is one of pathogenic and economic importance in domestic and wild ruminants, including the European bison, Bison bonasus Linnaeus (order Cetartiodactyla; family Bovidae); a species on the International Union for Conservation of Nature's Red List of Threatened Species. Carabus granulatus Linnaeus (order Coleoptera; family Carabidae) is one of the most prevalent species of ground beetle, inhabiting a wide range of terrestrial ecosystems in Poland. Twenty-six ground beetles of this species inhabiting the Bialowieza Primeval Forest in eastern Poland were screened for the presence of DNA of pathogenic gastrointestinal nematodes of ruminants. Extracted DNA was sequenced and compared to reference sequences. In six insects, the presence of H. contortus DNA was detected. The obtained nucleotide sequences were homologous to each other and to the majority of the published DNA sequences of H. contortus isolates. The sequences were also identical to a sequence of H. contortus isolated from European bison in Poland. The study provides the first molecular evidence of the presence of H. contortus DNA in C. granulatus. The finding suggests that ground beetles may play a role in the transmission dynamics of this parasite.

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.

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 New Haplotypes of Bartonella spp. and Borrelia burgdorferi Sensu Lato Identified in Lipoptena spp. (Diptera: Hippoboscidae) Collected in the Areas of North-Eastern Poland.

Deer keds are hematophagous ectoparasites (Diptera: Hippoboscidae) that mainly parasitize Cervidae. These flies are particularly important for animal health due to the occurrence of numerous pathogenic microorganisms. They may also attack humans and their bites may cause allergenic symptoms. The aim of the study was to identify the molecular characteristics of Borrelia burgdorferi sensu lato and Bartonella spp. pathogens detected in Lipoptena spp. sampled both from the hosts and from the environment. For identification of Bartonella spp and B. burgdorferi s. l., the primers specific to the rpoB and flaB gene fragments were used, respectively. The overall prevalence of B. burgdorferi s.l. DNA in Lipoptena cervi was 14.04%, including 14.8% infection in the tested group of winged specimens. The overall prevalence of Bartonella spp. was 57.02%. The presence of these bacteria was detected in 53.5% of specimens of L. cervi and 75.7% of L. fortisetosa. The phylogenetic analysis showed five new haplotypes of the rpoB gene of Bartonella sp. isolated from L. cervi/Lipoptena fortisetosa. We also identified one new haplotype of B. afzelii and three haplotypes of B. burgdorferi isolated from winged specimens of L. cervi. This is the first study to detect the genetic material of B. burgdorferi s.l. in L. cervi in Poland and the first report on the identification of these bacteria in host-seeking specimens in the environment.

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.

Ashworthius sidemi in cattle and wild ruminants in Poland - the current state of play.

Ashworthius sidemi, a blood-sucking abomasal nematode, has been identified in various wild ruminants, including deer (Cervus elaphus), roe deer (Capreolus capreolus), fallow deer (Dama dama) and moose (Alces alces). Although it has been observed throughout Poland, most sightings have been in the eastern part of the country. However, more recently, A. sidemi has been confirmed in the Ruszów Forest District (Lower Silesian Wilderness). It is now possible to test the faeces of cattle for the DNA of the third-stage infectious larvae (L3) of A. sidemi. The present paper describes such a molecular study of 120 faecal samples collected from cattle grazed in the Ruszów Forest District and Biebrza Marshland, where A. sidemi had previously been detected in wildlife. In this study, no A. sidemi DNA was identified in any of the examined samples.

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.

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.

Pathological lesions in the lungs of red deer Cervus elaphus (L.) induced by a newly-described Dictyocaulus cervi (Nematoda: Trichostrongyloidea).

The large lungworms of the genus Dictyocaulus are causative agents of parasitic bronchitis in various ungulate hosts, including red deer. Recently, the red deer-derived lungworm D. cervi was described and separated from D. eckerti. Little is known of the transmission patterns, epidemiology, geographical distribution and pathogenicity of D. cervi. Histological examinations were performed on 22 formalin-fixed lung tissue samples of hunted red deer. Exclusively, D. cervi adults were derived from 15 red deer and confirmed molecularly (GenBank accession: MH183394). Dictyocaulus cervi infection was associated with various degrees of lung pathology, including interstitial pneumonia, bronchitis and bronchiolitis with an influx of eosinophils, lymphocytes, plasma cells and macrophages; massive hyperplasia of lymphoid follicles within bronchiolar tissue, and hyperplasia of the bronchial and bronchiolar epithelium. Furthermore, emphysema, atelectasis and lung tissue congestion were noted. Interestingly, interstitial and subpleural fibrosis was seen in adult Dictyocaulus-negative samples, suggesting either a prepatent phase of Dictyocaulus infection or infection/coinfection with protostrongylid nematodes.

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.

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.

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.

Molecular surveillance of Theileria equi and Anaplasma phagocytophilum infections in horses from Ukraine, Poland and Slovakia.

A survey was undertaken to assess the prevalence of Theileria equi and Anaplasma phagocytophilum in some regions of Ukraine, Poland and Slovakia. Using a specific PCR assays, blood samples from 215 horses were tested. The prevalence of T. equi and A. phagocytophilum infection was 13.95% and 1.4%, respectively. BLAST analysis showed the isolates closest to the T. equi 18S rRNA and A. phagocytophilum msp4 gene sequences in GenBank with a similarity of ≥99%. No significant association was found between the T. equi PCR positivity and the age or sex of the horses. There was a significant association between the origin of horses and T. equi-PCR positivity. No significant association was found between the A. phagocytophilum-PCR positivity and the age, sex or origin.

The first detection of the tick-borne encephalitis virus (TBEV) RNA in Dermacentor reticulatus ticks collected from the lowland European bison (Bison bonasus bonasus L.).

Tick borne encephalitis virus (TBEV) (Flaviviridae, Flavivirus) is the causative agent of tick-borne encephalitis (TBE), a potentially fatal neurological infection. The disease is endemic in a large region in Eurasia, where is transmitted mainly by hard ticks: Ixodes ricinus and I. persulcatus. It is known that also Dermacentor reticulatus is involved in a circulation of TBEV, but the knowledge of its importance in the TBE epidemiology is still insufficient. The Bialowieza Primeval Forest is located in eastern Poland and it is a well-known endemic focus of tick-borne encephalitis. The aim of this study was to identify the prevalence of tick-borne encephalitis virus (TBEV) in Dermacentor reticulatus ticks collected from European bison (Bison bonasus bonasus), an important host of hard ticks in the Bialowieza Primeval Forest. In the years 2008-2009, a total of 114 adult D. reticulatus ticks were collected from 7 European bison and examined individually for the presence of TBEV RNA using nested RT-PCR assay. Positive results were noted in 18.42% of ticks. This is the first record of TBEV infection in ticks collected from European bison.