Cytokine gene polymorphism and parasite susceptibility in free-living rodents: Importance of non-coding variants.

Associations between genetic variants and susceptibility to infections have long been studied in free-living hosts so as to infer the contemporary evolutionary forces that shape the genetic polymorphisms of immunity genes. Despite extensive studies of proteins interacting with pathogen-derived ligands, such as MHC (major histocompatilbility complex) or TLR (Toll-like receptors), little is known about the efferent arm of the immune system. Cytokines are signalling molecules that trigger and modulate the immune response, acting as a crucial link between innate and adaptive immunity. In the present study we investigated how genetic variation in cytokines in bank voles Myodes glareolus affects their susceptibility to infection by parasites (nematodes: Aspiculuris tianjensis, Heligmosomum mixtum, Heligmosomoides glareoli) and microparasites (Cryptosporidium sp, Babesia microti, Bartonella sp.). We focused on three cytokines: tumour necrosis factor (TNF), lymphotoxin alpha (LTα), and interferon beta (IFNß1). Overall, we identified four single nucleotide polymorphisms (SNPs) associated with susceptibility to nematodes: two located in LTα and two in IFNß1. One of those variants was synonymous, another located in an intron. Each SNP associated with parasite load was located in or next to a codon under selection, three codons displayed signatures of positive selection, and one of purifying selection. Our results indicate that cytokines are prone to parasite-driven selection and that non-coding variants, although commonly disregarded in studies of the genetic background of host-parasite co-evolution, may play a role in susceptibility to infections in wild systems.

Long-term study of Borrelia and Babesia prevalence and co-infection in Ixodes ricinus and Dermacentor recticulatus ticks removed from humans in Poland, 2016-2019.

BACKGROUND: Lyme borreliosis (LB) is the most common vector-borne disease in Europe. Monitoring changes in the prevalence of different Borrelia species in ticks may be an important indicator of risk assessment and of differences in pathogenicity in humans. The objective of our study was to assess the prevalence, co-infection and distribution of Borrelia and Babesia species in ticks removed from humans in a large sample collected during a study period of 4 years. METHODS: The ticks were collected throughout Poland from March to November over 4-year period from 2016 to 2019. All ticks (n = 1953) were morphologically identified in terms of species and developmental stage. Molecular screening for Borrelia and Babesia by amplification of the flagellin gene (flaB) or 18S rRNA marker was performed. Pathogen identity was confirmed by Sanger sequencing or PCR-restriction fragment length polymorphism analysis. RESULTS: The ticks removed from humans in Poland during this study belonged to two species: Ixodes ricinus (97%) and Dermacentor reticulatus (3%). High Borrelia prevalence (25.3%), including B. miyamotoi (8.4%), was confirmed in Ixodes ricinus ticks removed from humans, as was the change in frequency of occurrence of Borrelia species during the 4-year study. Despite Babesia prevalence being relatively low (1.3%), the majority of tested isolates are considered to be pathogenic to humans. Babesia infection was observed more frequently among Borrelia-positive ticks (2.7%) than among ticks uninfected with Borrelia (0.8%). The most frequent dual co-infections were between Borrelia afzelii and Babesia microti. The presence of Borrelia was also confirmed in D. reticulatus (12.7%); however the role of these ticks in spirochete transmission to susceptible hosts is still unclear. CONCLUSIONS: Although the overall risk of developing LB after a tick bite is low in Europe, knowledge of the prevalence and distribution of Borrelia and Babesia species in ticks might be an important indicator of the risk of both these tick-borne diseases.

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.

Emerging risk of Dirofilaria spp. infection in Northeastern Europe: high prevalence of Dirofilaria repens in sled dog kennels from the Baltic countries.

Dirofilariasis is a fast-spreading disease of dogs and humans in Europe. We investigated whether Dirofilaria spp. have spread northwards in Europe, invading the Baltic countries. Altogether, 424 blood samples were collected from eight countries in the period 2017-2019, including 227 samples from sled dogs and 197 samples from other dogs. PCR amplification and sequencing were conducted employing three genetic markers (mitochondrial [mt] 12S rDNA, mt cytochrome c oxidase subunit I [COI] gene and mt dehydrogenase subunit I [NAD1] gene). The SNAP test (IDEXX) for detection of D. immitis infections was also implemented. The DNA of D. repens was detected in 59 of 424 dogs (prevalence 13.9%). D. repens was found in sled dogs from Lithuania, Latvia, Poland and Belarus. Only one dog from Estonia was infected, apparently an imported case. The highest prevalence was recorded in Lithuania (38%). Among pet dogs from the Ukraine, six dogs tested positive (3.8%). Our study has revealed a high prevalence of D. repens infections in Lithuania and Latvia, but no evidence for spread of the heartworm D. immitis. We conclude that sled dog kennels constitute hot spots for D. repens transmission.

The red fox (Vulpes vulpes), a possible reservoir of Babesia vulpes, B. canis and Hepatozoon canis and its association with the tick Dermacentor reticulatus occurrence.

The red fox (Vulpes vulpes) is known to be a reservoir host of various vector-borne protozoan parasites. Yet, the impact of these parasitic infections on the health status of the red fox is unknown. In this study, we compared the prevalence of haemoprotozoa in juvenile and adult foxes from four regions in Poland. The abundance of questing adult Dermacentor reticulatus ticks was shown to differ between regions and include a tick-endemic and non-endemic region, as well as two zones in which D. reticulatus recently expanded: an eastern and a western zone. Spleen and kidney enlargement indices were compared between infected, co-infected and non-infected foxes to evaluate pathogenic output of parasitic infections. Blood samples were collected from 383 necropsied red foxes and 25 live-trapped red fox cubs. The weights of spleens, kidneys and bodies were recorded for each necropsied individual. Blood and spleen DNA samples were screened by PCR to detect 18S rRNA and cox1 gene fragments of Babesia spp., and 18S rRNA of Hepatozoon spp. Prevalence of infection and mean organ weight indices were compared in relation to fox age and sex, origin (zone) and infection status. Hepatozoon canis (174/381; 45.7%) and Babesia vulpes (117/381; 30.7%) were the dominant pathogens infecting adult foxes. Babesia canis DNA was detected in 9 (2.4%) individuals. Two fox cubs from Western Poland were infected with B. vulpes (2/14; 14%), while H. canis infection (16/25; 59%) was detected in cubs from all three regions where trapping was carried out. Infection with B. vulpes was associated with significantly higher weights of spleens and kidneys. Spleen weight/ body weight and mean kidney weight/ body weight indices were shown as relevant in evaluating changes occurring during infection with B. vulpes. Babesia and Hepatozoon spp. infections differed in red foxes from D. reticulatus-endemic, non-endemic and newly inhabited areas. The prevalence of B. canis in foxes from four regions of Poland reflects the prevalence recorded in questing adult D. reticulatus collected in these areas. This finding suggests a contribution of red foxes to the establishment of new foci of B. canis-infected D. reticulatus ticks in areas recently invaded by this tick species. Spleen and kidney enlargement was identified in foxes infected with B. vulpes, particularly in individuals co-infected with B. vulpes and H. canis.

The Efficiency of Live-Capture Traps for the Study of Red Fox (Vulpes vulpes) Cubs: A Three-Year Study in Poland.

Safe and efficient techniques for the live capture of carnivores are limited. In this study, we identified some of the factors that could affect the success of capturing red fox cubs with live capture traps (also known as cage traps). During a three-year period, we analysed 32 captures of 25 fox cubs (1.3 captures/fox). We assessed the impact of the following factors: sex of animals, month of trapping, weather conditions recorded for each trap-night, the willingness of cubs to explore and enter cage traps, the researchers' activity around den complexes before trapping and distances to the nearest village or farm. The overall trap rate (32 captures, including recaptured cubs) and the trap rate for individual cubs (25 captures) was 11.2 cubs/100 trap-nights and 8.7/100 trap-nights, respectively. Animals other than foxes were captured only three times, thus the selectivity of the cage-trapping method was high (32/35 = 91.4%). The probability of capturing one cub per night was 70.2% (32 cubs/ 47 nights). Cubs inhabiting dens in the vicinity of human settlements were less likely to explore and enter traps. Vixens were more likely to relocate their litters if the activity of the staff setting the traps was intense at the trapping site. The success of trapping was higher during poor weather as, for example, during rain or thunderstorms. None of the trapped animals suffered any injuries. Whereas cage trapping can be an effective and safe capture method for juvenile foxes, capture efficiency is affected by the experience of the trappers and a range of other factors including weather and distance to human settlements.

Rodents as intermediate hosts of cestode parasites of mammalian carnivores and birds of prey in Poland, with the first data on the life-cycle of Mesocestoides melesi.

BACKGROUND: Rodents constitute an important part of the diet of many carnivore species. This predator-prey food chain is exploited by helminth parasites, such as cestodes, whose larval stages develop in rodents and then mature to the adult stage in predators. The main aim of our study was to use molecular techniques for identification of cestode species recovered from both intermediate and definitive hosts, with a particular focus on the genus Mesocestoides. METHODS: Larval cestodes were obtained during our long-term studies on rodent helminth communities in the Mazury Lake District in the north-east Poland in 2000-2018. Cestode larvae/cysts were collected from body cavities or internal organs (e.g. liver) during autopsies. Adult tapeworms were derived from nine red foxes, three Eurasian badgers and one Eurasian lynx. PCR amplification, sequencing and phylogenetic analyses were conducted employing three genetic markers: 18S rDNA, mitochondrial (mt) 12S rDNA and the mt cytochrome c oxydase subunit 1 (cox1) gene fragment. RESULTS: Altogether 19 Mesocestoides samples were analyzed, including 13 adult tapeworms from definitive hosts and six larval samples from 4 bank voles and 2 yellow-necked mice. Phylogenetic analyses revealed three well-supported trees of similar topology. In each case the Mesocestoides samples formed two separate clades. All isolates from foxes, the lynx isolate and two isolates from rodents grouped with Mesocestoides litteratus. Four isolates from rodents and all three isolates from Eurasian badgers were resolved in a separate clade, most similar to North American M. vogae (syn. M. corti). Examination of fixed, stained adult specimens from Eurasian badgers revealed consistency with the morphology of Mesocestoides melesi. Therefore, this clade is likely to represent M. melesi, a species first described in 1985 from the Eurasian badger Meles meles. Molecular analysis allowed also the identification of Taenia crassiceps, Hydatigera kamiyai and Cladotaenia globifera among larvae derived from rodents. CONCLUSIONS: Molecular and phylogenetic analyses support the recognition of M. melesi as a valid species. Our data represent the first record of the larvae of this species in rodents. This is the first report on the occurrence of H. kamiyai in rodents from Poland.

The role of juvenile Dermacentor reticulatus ticks as vectors of microorganisms and the problem of 'meal contamination'.

Juvenile Dermacentor reticulatus ticks inhabit nests and burrows of their rodent hosts and cannot be collected from vegetation. To detect vertical transmission of Babesia canis in D. reticulatus, we studied larvae and nymphs collected from rodents. However, the molecular techniques used for detection of pathogen DNA are sensitive enough to detect not only pathogens vectored by ticks but also those taken up with current or previous blood meals ('meal contamination') or just present in the environment and on the tick or host surface ('environmental contaminations'). Thus, an additional aim of our study was to evaluate the extent of such contamination while studying feeding ticks collected from rodents. Juvenile D. reticulatus were collected from 140 rodents: 91 bank voles trapped in two forest sites in the Mazury Lake District and 49 rodents (Apodemus and Microtus spp.) from an open habitat near the town of Bialobrzegi in Central Poland. Altogether 504 D. reticulatus ticks, comprising 266 individually evaluated nymphs and 238 larvae assigned to 50 larval pools, were studied for the presence of Babesia, Bartonella and Rickettsia spp. DNA. Statistical analyses were conducted to (1) evaluate the effect of rodent host factors (species, sex and age) on prevalence of infection in ticks, and (2) to compare the frequency of positive samples between groups of pathogen-positive and pathogen-negative rodent hosts. To complete the last aim, blood samples obtained from 49 rodents from Bialobrzegi were studied for the presence of Babesia and Bartonella DNA. Infestation of rodent hosts with juvenile ticks ranged between 46 and 78%, with a mean abundance of 3.6 ticks/rodent for D. reticulatus and 4.8 ticks/rodent for Ixodes ricinus. The highest prevalence of PCR-positive D. reticulatus samples was obtained for Rickettsia spp. (28%) and R. raoultii was identified in 22 sequenced PCR products. Babesia DNA was detected in 20 (7.5%), including B. microti in 18 (6.8%) and B. canis in two (0.8%) of 266 D. reticulatus nymphs that were analyzed. Babesia microti DNA was also detected in four pools of D. reticulatus larvae (4/50 pools = 8%). The detection success of B. microti in D. reticulatus was associated with the species of the rodent hosts of the ticks (much higher for typical B. microti-host-species such as Microtus spp. than for Apodemus spp.) and host age (3 × higher in ticks collected from adult hosts in comparison to juvenile ones). Moreover, the DNA of B. microti was detected in 68% of D. reticulatus nymphs collected from B. microti-positive rodents in comparison to only 1.6% of nymphs collected from B. microti-negative rodents. Bartonella DNA was detected in 18% of D. reticulatus tick samples (38% of larval pools, 14% of nymphs). Again, host factors played important roles for 'tick positivity'-the highest prevalence of positive ticks was on Apodemus spp., which are regarded as Bartonella reservoirs. Bartonella DNA was detected in 42% of nymphs and 57% of larval pools collected from Bartonella-positive rodents in comparison to 28% of nymphs and 11% of larvae collected from Bartonella-negative rodents. Vertical transmission of B. canis in D. reticulatus ticks was confirmed in the field. Additionally, we demonstrated that 'meal contamination' generates a confounding signal in molecular detection of pathogen DNA extracted from ticks collected from infected hosts and must be taken into account in evaluating the competence of tick species as vectors.

A new focus of the tick Haemaphysalis concinna in Western Poland.

The relict tick Haemaphysalis concinna has a fragmented and focal distribution in Central Europe and Asia. Although in the majority of neighboring countries the occurrence of this tick species is well-documented (i.e., in Germany, Czech Republic, Slovakia, Ukraine), to-date its occurrence in Poland has been registered only once, in 1953 in Troszyn in North-Western Pomerania, close to the German-Polish border. In the present study we report the first documented finding of H. concinna in Western Poland, confirmed both by collection of juvenile ticks from rodent hosts and questing ticks from vegetation. Trapping of rodents took place in the summer of 2018 in three locations in Western Poland (Slonin, Nowy Mlyn 1, Nowy Mlyn 2). Rodents were inspected for ectoparasites, which were detached and fixed in 70% ethanol. All the collected ticks were assigned to species and developmental stages using appropriate morphological keys, and representative individuals were genotyped by molecular methods. A total of 1482 feeding ticks were collected from 106 rodents from three sites. The common tick Ixodes ricinus was found in abundance on small rodents at all three sites; Dermacentor reticulatus ticks were identified at two sites in small numbers and, finally, numerous juvenile H. concinna (n = 427) were found at one of our study sites (Nowy Mlyn 2). The highest prevalence and abundance of H. concinna were recorded on voles, Microtus agrestis and M. oeconomus, from this site in August. Additionally, questing nymphs and adult H. concinna were collected locally from vegetation (n = 20). Genotyping and phylogenetic analysis confirmed the species as H. concinna. A new focus of H. concinna has been described in Western Poland. Our long-term field work monitoring the expansion of the distribution of D. reticulatus in Poland, during which all collected ticks are identified, suggests that H. concinna is still very rare in the country.

Long-term spatiotemporal stability and dynamic changes in helminth infracommunities of spiny mice (Acomys dimidiatus) in St. Katherine's Protectorate, Sinai, Egypt.

The importance of parasites as a selective force in host evolution is a topic of current interest. However, short-term ecological studies of host-parasite systems, on which such studies are usually based, provide only snap-shots of what may be dynamic systems. We report here on four surveys, carried out over a period of 12 years, of helminths of spiny mice (Acomys dimidiatus), the numerically dominant rodents inhabiting dry montane wadis in the Sinai Peninsula. With host age (age-dependent effects on prevalence and abundance were prominent) and sex (female bias in abundance in helminth diversity and in several taxa including Cestoda) taken into consideration, we focus on the relative importance of temporal and spatial effects on helminth infracommunities. We show that site of capture is the major determinant of prevalence and abundance of species (and higher taxa) contributing to helminth community structure, the only exceptions being Streptopharaus spp. and Dentostomella kuntzi. We provide evidence that most (notably the Spiruroidea, Protospirura muricola, Mastophorus muris and Gongylonema aegypti, but with exceptions among the Oxyuroidae, e.g. Syphacia minuta), show elements of temporal-site stability, with a rank order of measures among sites remaining similar over successive surveys. Hence, there are some elements of predictability in these systems.

Abundance of the tick Dermacentor reticulatus in an ecosystem of abandoned meadows: Experimental intervention and the critical importance of mowing.

The effect of agricultural activities on the environment has been falling in many areas of Europe in recent years and the associated abandonment of crop fields, meadows and pastures may enable an increase in tick densities. In the present study we assessed whether regular mowing would have a negative effect on Dermacentor reticulatus populations and whether the cessation of regular mowing would cause an increase in abundance of D. reticulatus ticks. Two field experiments were conducted during a five-year period (2012-2016) in the Mazowieckie (Mazovia) region of Central Poland. Experiment 1: The long-term effect of mowing on tick population was tested in the meadow ecosystem of Stoski, an old fallow land plot that was mowed three times a year. Experiment 2: Neglecting the cultivation by abandonment of arable land was evaluated in Kury village. Four areas (2 experimental 'fallow lands', 2 control meadows) were selected. The first fallow land plot was a fenced off area comprising mostly of a horse pasture and the second fallow land plot was designated in an old abandoned orchard. At each site, ticks were collected in consecutive springs and autumns by dragging at least twice during each season from experimental and control areas. Altogether 1452 D. reticulatus ticks were collected and their densities were compared by multifactorial ANOVA. In the end of the first experiment, a significant decrease (6 times) in tick abundance was observed in the mowed area in comparison to old fallow land. In the end of the second experiment, tick abundance was three times higher in the experimental fallow lands in comparison to the control meadows. In conclusion it was found that regular mowing significantly reduced the density of questing D. reticulatus ticks in open areas. Cessation of mowing may enhance the number of ticks and the associated risk of acquiring tick-borne diseases.

Genetic and phylogenetic analysis of the ticks from the Sinai Massif, Egypt, and their possible role in the transmission of Babesia behnkei.

Following the description of Babesia behnkei in the region of St. Katherine, Sinai, the present study was undertaken to determine the role of local tick species as vectors of piroplasms. First we assessed the local fauna of ticks, especially species occurring on rodents, camels and encountered in the environment, and then we compared genotypes of ticks from isolated wadis. Finally, we assessed the role of local tick species as potential vectors of Babesia spp. During our expedition to the Sinai Massif in a 4-week period in August-September 2012, 393 ticks were collected, including 235 adult questing ticks collected from the environment (ground level in the wadis) and 158 engorging ticks from camels and rodents. Amplification and sequencing of a 600 bp fragment of the conservative 18S rDNA and a 440 bp fragment of the more variable mitochondrial (mt) 16S rDNA were carried out to enable the identification of 54 ticks and to assess the genetic variability of ticks collected from two distant isolated wadis. The camel tick Hyalomma dromedarii constituted the majority (80-90%) of adult ticks collected from three wadis in the Sinai Mountains near St. Katherine. Among juvenile ticks collected from rodents, three genotypes were identified: H. dromedarii; Hyalomma sp. showing low homology with H. dromedarii, H. lusitanicum or H. aegyptium; and Rhipicephalus sp. A new genotype of Hyalomma was identified in an isolated montane valley, W. Gebal. Babesia/Theileria DNA was not detected in any of the ticks, which is likely due to the low infection rate in the limited number of ticks that were examined.

Spread of Dermacentor reticulatus is associated with the loss of forest area.

Changes in tick distribution and abundance are among the main drivers of the (re)emergence of transmitted pathogens. We aimed to uncover the reasons of the reported spread of Dermacentor reticulatus in Poland using a variety of proxies of environmental features, ground-measured temperature and remotely-sensed data of temperature and vegetation. Ground-measured temperature was recorded in 2013-2014, in sites where D. reticulatus presence (n = 16) or absence (n = 16) was confirmed. We specifically aimed to test whether changes in phenology of vegetation and the length of the growing season were correlated with field-derived data regarding the presence/absence of D. reticulatus. We also used categorical descriptions of the habitat to capture the vegetation patterns that might delineate the distribution of the tick. We demonstrated that temperature, phenology of vegetation and length of growing season have no correlation with the occurrence of D. reticulatus in Poland. There is, however, a clear association between the trends of the loss of forests and the presence of the tick. This parameter was two times higher at sites colonized by D. reticulatus in comparison to the sites where the population of the tick is not yet established. A spatial analysis demonstrated that the preferred territories for D. reticulatus are those of a highly fragmented landscape within a large patch of homogeneous vegetation, in the vicinity of permanent water courses or reservoirs.

Description of Candidatus Bartonella fadhilae n. sp. and Candidatus Bartonella sanaae n. sp. (Bartonellaceae) from Dipodillus dasyurus and Sekeetamys calurus (Gerbillinae) from the Sinai Massif (Egypt).

Bartonella spp. are parasites of mammalian erythrocytes and endothelial cells, transmitted by blood-feeding arthropod ectoparasites. Different species of rodents may constitute the main hosts of Bartonella, including several zoonotic species of Bartonella. The aim of this study was to identify and compare Bartonella species and genotypes isolated from rodent hosts from the South Sinai, Egypt. Prevalence of Bartonella infection was assessed in rodents (837 Acomys dimidiatus, 73 Acomys russatus, 111 Dipodillus dasyurus, and 65 Sekeetamys calurus) trapped in 2000, 2004, 2008, and 2012 in four dry montane wadis around St. Katherine town in the Sinai Mountains. Total DNA was extracted from blood samples, and PCR amplification and sequencing of the Bartonella-specific 860-bp gene fragment of rpoB and the 810-bp gene fragment of gltA were used for molecular and phylogenetic analyses. The overall prevalence of Bartonella in rodents was 7.2%. Prevalence differed between host species, being 30.6%, 10.8%, 9.6%, and 3.6% in D. dasyurus, S. calurus, A. russatus, and A. dimidiatus, respectively. The phylogenetic analyses of six samples of Bartonella (five from D. dasyurus and one from S. calurus) based on a fragment of the rpoB gene, revealed the existence of two distinct genetic groups (with 95-96% reciprocal sequence identity), clustering with several unidentified isolates obtained earlier from the same rodent species, and distant from species that have already been described (90-92% of sequence identity to the closest match from the GenBank reference database). Thus, molecular and phylogenetic analyses led to the description of two species: Candidatus Bartonella fadhilae n. sp. and Candidatus Bartonella sanaae n. sp. The identification of their vectors and the medical significance of these species need further investigation.

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 prevalence of Dirofilaria repens in cats, healthy dogs and dogs with concurrent babesiosis in an expansion zone in central Europe.

BACKGROUND: Dirofilaria repens is a mosquito-transmitted, filarial nematode parasitizing dogs, cats and other carnivores. Recently, this parasite has spread in central Europe, including Poland. The aim of the present study was to estimate the prevalence of D. repens in cats and dogs in different regions of the country and to investigate the occurrence and consequences of co-infection with another fast-spreading vector-borne parasite, Babesia canis. RESULTS: In the period 2013-2015, 147 blood samples from cats from central Poland and 257 blood samples from dogs from central, northern, southern and western Poland were collected. Prevalence of D. repens was determined by amplification and sequencing of the 12S rDNA gene fragment. Among dogs, 94 samples originated from clinically healthy dogs from central Poland (Masovia) and 58 samples originated from dogs that were infected with B. canis. Prevalence of D. repens was compared between these two groups of dogs. For the first time D. repens was identified in a cat from central Europe (0.7 % [95 % CL: 0-4.1 %]). The DNA of the filarial endosymbiotic bacterium Wolbachia was detected in two cats (1.4 % [95 % CL: 0-5.5 %]). In dogs, the parasite was detected only in samples from central Poland (Masovia) (local prevalence = 38 % [95 % CL: 25.9-51.8 %]). Prevalence of D. repens was significantly higher in dogs with babesiosis (90 % [95 % CL: 81.6-94.5 %]). Co-infections of D. repens and B. canis were confirmed by sequencing in 30 dogs with babesiosis, but no co-infections were identified in healthy dogs from Masovia. Statistical analyses of blood parameters revealed that dogs with co-infections suffered more severe anemia and thrombocytopenia, but presented milder changes in biochemical parameters (i.e. less elevated concentration of alkaline phosphatase [ALP] and serum urea) suggesting lower risk of hepatic or renal failure in comparison to dogs infected only with B. canis. CONCLUSIONS: These findings are important due to the spread of dirofilariosis and babesiosis in central Europe, as microfilaraemic dogs seem to be more prone to babesiosis. The possible protective effect of the nematode infection against hepatic or renal failure in canine babesiosis and its mechanisms require further investigations.

Long-term spatiotemporal stability and dynamic changes in the haemoparasite community of spiny mice (Acomys dimidiatus) in four montane wadis in the St. Katherine Protectorate, Sinai, Egypt.

BACKGROUND: Long-term field studies of parasite communities are rare but provide a powerful insight into the ecological processes shaping host-parasite interactions. The aim of our study was to monitor long-term trends in the haemoparasite communities of spiny mice (Acomys dimidiatus) and to identify the principal factors responsible for changes over a 12 year period. METHODS: To this end we sampled four semi-isolated populations of mice (n = 835) in 2000, 2004, 2008 and 2012 in four dry montane valleys (wadis) located in the Sinai Massif, Egypt. RESULTS: Overall 76.2 % of spiny mice carried at least one of the five haemoparasite genera (Babesia, Bartonella, Haemobartonella, Hepatozoon, Trypanosoma) recorded in the study. Prevalence of haemoparasites varied significantly between the sites with the highest overall prevalence in Wadi Tlah and the lowest in W. El Arbaein, and this changed significantly with time. In the first two surveys there was little change in prevalence, but by 2008, when the first signs of a deepening drought in the region had become apparent, prevalence began to drift downwards, and by 2012 prevalence had fallen to the lowest values recorded from all four sites over the entire 12-year period. The overall mean species richness was 1.2 ± 0.03, which peaked in 2004 and then dropped by more than 50 % by 2012. Species richness was highest among mice from Wadi Tlah and peaked in age class 2 mice (young adults). Site was the most significant factor affecting the prevalence of individual parasite species, with Trypanosoma acomys and Hepatozoon sp. occurring mainly in two wadis (W. Tlah & W. Gharaba). In four of the five genera recorded in the study we observed a significant drop in prevalence or/and abundance since 2004, the exception being Hepatozoon sp. CONCLUSIONS: During the 12-year-long period of study in the Sinai, we observed dynamic changes and possibly even cycles of prevalence and abundance of infections which differed depending on parasite species. Although the exact reasons cannot be identified at this time, we hypothesize that the effects of a 15-year-long scarcity of rainfall in the local environment and a fall in host densities over the period of study may have been responsible for a drop in transmission rates, possibly by a negative impact on vector survival.

Mapping of Dermacentor reticulatus expansion in Poland in 2012-2014.

Rapid expansion of the tick Dermacentor reticulatus (Fabricius) has been reported in many European countries. In Poland its range was limited to the area on the eastern side of the Vistula River up until the 1990s. However, new foci were recently discovered, while the centre of the country and mountain regions are believed to be free of this tick and are known as "the gap". A few unconfirmed factors, like unfavourable weather conditions, changes in land cover and absence of competent hosts have previously been reported as possible reasons for the absence of D. reticulatus in this area. Since D. reticulatus plays an important role for the maintenance and the circulation of tick-borne pathogens, we (1) determined its actual range in Poland, (2) monitored its expansion in 2012-2014 and (3) correlated abiotic conditions on its known range. Dragging was conducted in the area between the Vistula River and the western border of Poland in 2012-2014, along the three major Polish rivers and their tributaries. Temperature and humidity on the ground were recorded 4 times a day at a total of 32 sites. D. reticulatus was found in 21 new locations on the western side of the Vistula River and in 22 locations in western Poland. The presence of this tick species was confirmed for the first time in Wielkopolskie, Kujawsko-Pomorskie and Lódzkie voivodeships. Existence of 'the gap' was confirmed on a strip of land along the northwest-southeast axis (an area of approximately 151000km(2)), that extends between Zachodniopomorskie and Pomorskie in the North, and the mountain areas with foothills in the South, in Opolskie, Slaskie, Malopolskie and Podkarpackie voivodeships. This gap divides the tick population in Poland into two separated populations - Western and Eastern. The lowest abundance of ticks was found in the newly established foci in central and western Poland. Our study showed the need for the monitoring of the D. reticulatus expansion. Early detection of new foci is crucial for taking proper prevention measures.

Vertical Transmission of Babesia microti in BALB/c Mice: Preliminary Report.

Babesia spp. (Apicomplexa, Piroplasmida) are obligate parasites of many species of mammals, causing a malaria-like infection- babesiosis. Three routes of Babesia infection have been recognized to date. The main route is by a tick bite, the second is via blood transfusion. The third, vertical route of infection is poorly recognized and understood. Our study focused on vertical transmission of B. microti in a well-established mouse model. We assessed the success of this route of infection in BALB/c mice with acute and chronic infections of B. microti. In experimental groups, females were mated on the 1st day of Babesia infection (Group G0); on the 28th day post infection (dpi) in the post- acute phase of the parasite infection (G28); and on the 90th and 150th dpi (G90 and G150 group, respectively), in the chronic phase of the parasite infection. Pups were obtained from 58% of females mated in the post-acute phase (G28) and from 33% of females in groups G90 and G150. Mice mated in the pre-acute phase of infection (G0) did not deliver pups. Congenital B. microti infections were detected by PCR amplification of Babesia 18S rDNA in almost all pups (96%) from the experimental groups G28, G90 and G150. Parasitaemia in the F1 generation was low and varied between 0.01-0.001%. Vertical transmission of B. microti was demonstrated for the first time in BALB/c mice.

Pathogens vectored by the tick, Dermacentor reticulatus, in endemic regions and zones of expansion in Poland.

BACKGROUND: Dermacentor reticulatus plays an important role in the maintenance of pathogens of medical and veterinary importance in the environment. Currently two isolated populations of D. reticulatus are present in Poland--Western and Eastern. The range of the Eastern population covers endemic areas in eastern Poland but this population is expanding westwards creating an expansion zone in the centre of the country. The expansion zone in western Poland is occupied by the recently discovered Western population, spreading eastwards. METHODS: Questing adult ticks (n = 2585) were collected in 2012-2014 in endemic regions of north-eastern (Warminsko-Mazurskie Voivodeship) and central Poland (Masovian Voivodeship) and in the expansion zones in central and western Poland, in the region between the Vistula River and the western border of the country. Amplification of Babesia, Rickettsia spp. and Borrelia burgdorferi sensu lato DNAs was performed using specific starters. RNA of the TBE virus was detected using RT-PCR and representative PCR products were sequenced and compared with sequences deposited in GenBank. RESULTS: Of the total 2585 examined ticks, 1197 (46.3 %) were infected with at least one pathogen. Overall prevalence of pathogens was 4.18 % (108/2585) for Babesia spp., 44.10 % (1140/2585) for Rickettsia spp., 0.09 % (1/1107) for Borrelia afzelii and 7.6 % (7/92) for TBEV. Sequence analysis of DNA showed 99.86 % similarity to R. raoulti and 99.81 % to B. canis. One male from north-eastern Poland was infected with B. microti. Prevalence of R. raoulti was highest in the Western population (52.03 %) and lowest in the Eastern population in north-eastern Poland (34.18 %). Babesia canis was not detected in 592 ticks collected in the Western population, while in the Eastern population overall prevalence was 5.42 %. There were significant differences in the prevalence of B. canis between tick samples from northern (0.68 %), central (1.18 %) and southern (14.8 %) areas of the expansion zone in central Poland. CONCLUSIONS: Our study found significant differences between the range and prevalence of vectored pathogens in D. reticulatus from the endemic areas and newly inhabited expansion zones. The differences were likely associated with the different time of settlement or 'source' of ticks populations, the Eastern and the Western one.