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The European red squirrel (Sciurus vulgaris) is a common host for Ixodes ricinus ticks in urban and rural habitats, however, studies on ticks and tick-borne pathogens (TBPs) of squirrels have not been conducted in Poland yet. Thus, the aims of the current study were to assess and compare the prevalence and abundance of ticks on red squirrels trapped at two sites in the Warsaw area (in an urban forest reserve and an urban park) and using molecular tools, to assess the genetic diversity of three pathogens (Borrelia burgdorferi sensu lato, Rickettsia and Babesia spp.) in I. ricinus ticks collected from squirrels. For the detection of Rickettsia spp. a 750 bp long fragment of the citrate synthase gltA gene was amplified; for B. burgdorferi s.l. 132f/905r and 220f/824r primers were used to amplify the bacterial flaB gene fragments (774 and 605 bp, respectively) and for Babesia spp., a 550 bpfragment of 18S rRNA gene was amplified. In total, 91 red squirrels were examined for ticks. There were differences in tick prevalence and mean abundance of infestation in squirrels from the urban forest reserve and urban park. Three species of B. burgdorferi s.l., Rickettsia spp., and Babesia microti were detected in ticks removed from the squirrels. Our results broaden knowledge of S. vulgaris as an important host for immature I. ricinus stages and support the hypothesis that red squirrels act as a reservoir of B. burgdorferi. Moreover, we conclude that red squirrels may also play a role in facilitating the circulation of other pathogens causing serious risk of tick-borne diseases in natural and urban areas., (© 2024. The Author(s).)

Babesia canis, a widely distributed European tick-borne protozoan haemoparasite, causes canine babesiosis, the most important tick-borne disease afflicting dogs worldwide. The meadow tick, Dermacentor reticulatus, is considered to be the primary vector of this parasite in central Europe. Females of the more broadly distributed and medically important castor bean tick, Ixodes ricinus, also commonly feed upon dogs, but their role in the enzootic transmission cycle of B. canis is unclear. Here, we screened 1,598 host-seeking I. ricinus ticks collected from two different ecosystems, forest stands vs. urban recreational forests, for the presence of B. canis DNA. Ticks were sampled during their two seasonal peaks of activity, spring (May/June) and late summer (September). Babesia species were identified by amplification and sequencing of a hypervariable 18S rRNA gene fragment. Babesia canis was the only piroplasm detected in 13% of 200 larvae and 8.2% of 324 nymphs in the forest ecosystems. In urban recreational areas, B. canis DNA was found in 1.5% of 460 nymphs, 3.5% of 289 females and 3.2% of 280 males. Additionally, three samples, including one female, one male, and one nymph, were co-infected with B. venatorum and one nymph with B. divergens or B. capreoli. Our findings implicate that B. canis can be transmitted transovarially and maintained transstadially within populations of I. ricinus, but the vector competence of I. ricinus for transmitting B. canis remains to be investigated., (Copyright © 2021 The Authors. Published by Elsevier GmbH.. All rights reserved.)

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.

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

The 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., (Copyright © 2020 Elsevier GmbH. All rights reserved.)

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 Białobrzegi 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 Białobrzegi 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.

The objective of our study was to estimate the seroprevalence of six pathogens transmitted by ticks in HIV-infected persons and blood donors in Poland (B. burgdorferi s.l., A. phagocytophilum, Ehrlichia spp., Babesia spp., Rickettsia spp. Bartonella henselae) to assess the frequency of exposure to such microorganisms in immunocompetent and immunocompromised individuals in endemic regions for I. ricinus ticks. Serum samples were collected from 227 HIV-infected patients and 199 blood donors. All samples were analyzed for antibodies against six tick-borne pathogens and seroprevalence rates were statistically compared between two tested group as well as age, sex and lymphocyte T CD4+ level in HIV infected patients. The seroprevalence of tick-borne infections in HIV-infected patients is higher than that of the healthy population in Poland, although no association between serological status of patients and lymphocyte CD4+ T cell level has been observed. The frequency of tick-borne coinfections and doubtful results of serological tests were significantly higher in HIV-positive individuals. In Poland, the possibility of tick-borne diseases transmission with blood is rather negligible.

Objective: Bovine piroplasmoses are tick-borne protozoan diseases caused by parasites of the genus Theileria and Babesia . The aim of this work was to study the epizootic situation of babesiosis in the cattle population in eastern Poland and possibly to determine which species of protozoa infects Polish cattle., Material, Methods and Results: Blood samples for molecular analysis (real time PCR) were collected from 192 dairy cattle from various farms located in eastern Poland. The infection was detected in 10.4 % of the samples. All animals were infected with Babesia occultans which sequence of the 18S RNA gene fragment showed a 92.8 % homology with the sequence of B. occultans EU 376017., Conclusion and Clinical Relevance: This is the first report about the detection of B. occultans DNA in asymptomatic cattle in eastern Poland. The results obtained indicate that the range of these parasites is increasing and that continuous monitoring of babesiosis in cattle in Europe and in Poland is necessary., Competing Interests: The authors declare that they have no competing interests. The authors certify that they have no affiliation with or financial involvement in any organization or entity with a direct financial interest in the subject matter or the materials discussed in the manuscript., (Georg Thieme Verlag KG Stuttgart · New York.)

The aim of the paper was to study the epizootic situation of babesiosis in the cattle population in eastern Poland and possibly to determine what species of protozoa infects Polish cattle. Blood samples for molecular analysis (real time PCR) were collected from 192 dairy cows from various farms located in eastern Poland. The infection was detected in 10.4% of the samples. All animals were infected with Babesia occultans which sequence of the 18S RNA gene fragment showed a 93.1%, homology with the sequence of B. occultans EU 376017. This is the first report about the detection of B. occultans DNA in asymptomatic cattle in eastern Poland., (Copyright© by the Polish Academy of Sciences.)

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 (Warmińsko-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.

Anaplasma phagocytophilum and Babesia spp. are emerging tick-borne pathogens which can threaten human health. A duplex real-time PCR and qPCRs with primers and probes targeting 97 and 116 bp fragments of 16S rRNA and 18S rRNA genes, respectively, were used for qualitative and quantitative detection of both pathogens in Ixodes ricinus ticks. Altogether 1875 ticks (1084 adults and 791 nymphs) were collected from rural and urban habitats in northern Poland. Of them, at least 0.9% were found to be infected with A. phagocytophilum while 2.5% with Babesia spp. A comparison of the infection rates by the tick stage, the type of area, the collection site, habitats of different tick density and by the month of collection was done. The prevalence of pathogens was significantly lower in nymphs than in adult ticks (p = 0.02) and in rural areas than in urban areas (p = 0.007). Four different 16S rRNA gene variants of A. phagocytophilum were determine, however none of them showed 100% identity with compared sequences isolated from human patients. The dominant Babesia species was B. venatorum. Results of qPCRs with circular and linearized forms of plasmids used as the standards showed significant difference in the pathogen loads (p = 0.001). The copy numbers of A. phagocytophilum and Babesia spp. estimated from the linear plasmids were 28.7 and 5.1 times lower, respectively, when compared with their circular forms, and were accepted as more reliable. The average number of copies of 16S rRNA gene of A. phagocytophilum in the positive I. ricinus samples were 3.39 × 10(5) ± 6.09 × 10(5). The mean copy number of 18S rRNA gene of Babesia spp. was ~2.55 × 10(5) ± 1.04 × 10(6). We confirmed the presence of A. phagocytophilum and Babesia spp. in I. ricinus in both rural and urban environments. The determined low infection rates suggests, however, that the risk for local population and tourists to acquire infection is also low. Moreover, we confirmed recent findings that serious overestimation by circular plasmid DNA makes it less suitable as a standard and that the linear standards should be recommended for qPCR.

Introduction and Objective: Tick-borne infections constitute an increasing health problem in dogs and may lead to death, especially in young or elderly individuals. Canine babesiosis constitutes a serious health problem in dogs worldwide. The aim of the study was to verify the probability of vertical transmission of Babesia canis between the bitch and the pups., Materials and Methods: In Autumn 2011, cases of babesiosis were diagnosed in a litter of 6-week-old puppies of a Central Asian Shepherd dog. Immediately following the first case of infection, blood samples were collected from all the pups in the litter (n=10) and from the female. Detection of Babesia infection was performed by molecular and microscopical techniques., Results: The presence of B. canis DNA was detected using PCR in three pups, presenting at the time or 24-48 hours later with babesiosis symptoms, and in their asymptomatic mother. The isolates derived from the pups and the female - 520 bp 18S rRNA gene fragment - were compared and analyzed. All isolates from the pups and their mother were identical and showed 100% homology with B. canis group B (EU622793), supporting the same source of infection. Additionally, the USG of the peritoneal cavity was performed in the female, presenting evidence for splenomegaly., Conclusions: On the basis of (1) the same timing of three pup cases; (2) the identical B. canis sequences derived from all positive dogs; (3) evident splenomegaly in the asymptomatic female, this provides the first evidence of the vertical transmission of this piroplasm in dogs.

Wild species are essential hosts for maintaining Ixodes ticks and the tick-borne diseases. The aim of our study was to estimate the prevalence, the rate of co-infection with Babesia, Bartonella, and Anaplasma phagocytophilum, and the molecular diversity of tick-borne pathogens in roe deer in Poland. Almost half of the tested samples provided evidence of infection with at least 1 species. A. phagocytophilum (37.3%) was the most common and Bartonella (13.4%) the rarest infection. A total of 18.3% of all positive samples from roe deer were infected with at least 2 pathogens, and one-third of those were co-infected with A. phagocytophilum, Bartonella, and Babesia species. On the basis of multilocus molecular studies we conclude that: (1) Two different genetic variants of A. phagocytophilum, zoonotic and nonzoonotic, are widely distributed in Polish roe deer population; (2) the roe deer is the host for zoonotic Babesia (Bab. venatorum, Bab. divergens), closely related or identical with strains/species found in humans; (3) our Bab. capreoli and Bab. divergens isolates differed from reported genotypes at 2 conserved base positions, i.e., positions 631 and 663; and (4) this is the first description of Bart. schoenbuchensis infections in roe deer in Poland. We present 1 of the first complex epidemiological studies on the prevalence of Babesia, Bartonella, and A. phagocytophilum in naturally infected populations of roe deer. These game animals clearly have an important role as reservoir hosts of tick-borne pathogens, but the pathogenicity and zoonotic potential of the parasite genotypes hosted by roe deer requires further detailed investigation.

Introduction and Objectives: Sporting dogs, including sled dogs, are particularly prone to tick-borne infection either due to training/racing in forest areas or through visits to endemic areas. The aim was to present tick-borne infections in a 6-dog racing team after a race in Estonia., Materials and Methods: On the 4th day after return to Poland, the first dog presented with babesiosis symptoms and was diagnosed and treated accordingly. Next morning, the dog showed neurological symptoms and was diagnosed with tick-borne encephalitis (TBE). Diagnosis was confirmed by a high level of IgG antibodies (922 IU/ml), detected in serum 3 months later. The second dog presented with babesiosis symptoms on the 7th day after return. Babesia DNA was extracted from blood, amplified and sequenced to answer the question of whether the dogs became infected during the race in Estonia or in Poland., Results and Conclusions: Sequencing of a fragment of Babesia 18S rDNA revealed that these two isolates were identical to one another and closely related to the B. canis sequence originally isolated from the dog and Dermacentor reticulatus ticks in Poland. Thus, this is the first confirmed case of B.canis and TBEV co-infection and first confirmed case of TBE in a dog in Poland.

The aim of this paper was to present the first case of cerebral canine babesiosis due to infection by Babesia canis in a dog in Poland. A 5-year-old American Staffordshire Terrier was presented with an unusual clinical manifestation of acute babesiosis that included neurological signs and pancytopenia. Despite treatment the dog died. Diagnosis was based on microscopic examination of Giemsa-stained blood smears (detection of piroplasms in red blood cells) and post mortem examination of the brain by histopathology and PCR method. The amplified segment of the Babesia 18S RNA gene was sequenced. This enabled to determine that the cause of the disease had been the strain 18S RNA-B EU622793. This is one of two B. canis strains found endemically in Poland, which reveals a greater virulence than the strain 18S RNA-A EU622792. The described case indicates that this form of canine babesiosis should be taken into account in differential diagnosis in dogs exhibiting neurological symptoms, especially in the tick activity season.

The aim of this study was to perform in vitro cultivation of Babesia canis protozoa isolated from dogs with clinical babesiosis. A primary culture initiated in RPMI-1640 medium supplemented with 40% canine serum supported parasite growth in vitro in 5% carbon dioxide in air atmosphere. Subsequent subcultures into HL-1 medium with 40% dog serum or EMEM with 40% foetal bovine serum also supported parasite propagation. The parasites have been continuously cultured through six passages, although the parasitemias are low, ranging from 0.56% to 0.59%. The partial small subunit ribosomal rRNA gene sequence was identical in blood-derived and culture-derived Babesia. The parasites from 17 cultures were classified as EU622792, and from 13 cultures as EU622793. These data show that an efficient in vitro cultivation of B. canis could serve as a starting point to obtain a protozoan antigen used for immunisation of the dogs against piroplasmosis.

The aim of this study was to determine the usefulness of the real-time polymerised chain reaction (PCR) high-resolution melting (HRM) method in the differentiation of the Babesia canis canis protozoa isolated from dogs in the areas of eastern Poland. The studies involved 20 isolates of B. canis canis qualified depending on the analysis of the 18S RNA gene sequence to group A (EU 622792) and 20 isolates qualified to group B (EU 622793). It was proven with the real-time PCR technique that the melting temperature (Tm) of the obtained products of amplification was 78 degrees C for the representatives of group A and 81 degrees C for the representatives of group B, which proves that the real-time SYBR Green HRM PCR method is a technique allowing for the differentiation of the B. canis isolates which are slightly different with respect to the genetic structure, without the necessity to carry out time-consuming studies, i.e., sequencing and restriction fragment length polymorphism.

Co-infection with Borrelia species and Anaplasma phagocytophilum or Babesia spp. was assessed in a retrospective study of tick-exposed individuals from southeastern Poland. The co-infection rate of these pathogens was found to be rather low (Borrelia spp./Anaplasma phagocytophilum--4.2%, 1/24; Borrelia spp./Babesia spp.--4.2%, 1/24). However, due to the increased prevalence of Borrelia spp. in Ixodes ricinus ticks in Poland and the recent emergence of new tick-borne infections, it is necessary to carefully evaluate the true risk of human infection with several pathogens using more sensitive and reliable diagnostic tools. This is the first report of human infection with Babesia spp. in Poland that has been confirmed by molecular techniques with homology of 98.9% to B. divergens or Babesia EU1.

Each month, from March 2003 to February 2004, 34 blood samples from dogs were randomly selected from the blood samples delivered to two veterinary laboratories in Warsaw and tested for the DNA of Borrelia burgdorferi sensu lato, Anaplasma phagocytophilum, Babesia canis and Hepatozoon canis. Borrelia DNA was detected in seven of the 408 dogs, A phagocytophilum DNA was found in two, and B canis DNA was found in 48 (11.8 per cent). The DNA of H canis was not found in any of the blood samples. Sequencing of the seven Borrelia amplicons showed that only the genospecies Borrelia afzelii was present, the first time it has been detected in dogs in Poland.

The purpose of this review was to describe and discuss the current spectrum of coinfections in Ixodidae ticks and their effects on the transmission of blood microparasites. Coinfections with Borrelia burgdorferi s. l. and Anaplasma phagocytophilum and/or Babesia sp. in ticks from Poland appear to be common, however, the potential influence on transmission dynamics, the mechanism of genetic variation and the ecology of interactions between pathogens remain poorly understood compared with infections by single pathogen.

Ixodes ricinus, the most commonly observed tick species in Poland, is a known vector of such pathogenic microorganisms as TBE viruses, Borrelia burgdoferi sensu lato, Anaplasma phagocytophilum, Rickettsia helvetica, Babesia divergens and B. microti in our country. Our study aimed to find out whether this tick can also transmit other babesiae of medical and veterinary importance. DNA extracts of 1392 ticks (314 nymphs, 552 male and 526 female ticks) collected in urban and rural areas in the Pomerania province (northern Poland), were examined by nested PCR for the detection of Babesia spp., using outer primers: 5-22F and 1661R, and inner primers: 455-479F and 793-772R, targeting specific fragment of 18S rRNA gene. Overall, at least 1.6% ticks were found to be infected with babesial parasites. In the case of nymphs, the minimal prevalence was 0.6%, and it was approx. 3-times lower than in adults (1.9%). Percentages of infected males and females were comparable (2.0% vs. 1.7%). Sequences of 15/22 PCR-derived fragments of 18S rRNA gene demonstrated 100% similarities with the sequence of Babesia EUI (proposed name B. venatorum) (acc. no. AY046575) (n = 13) and with B. canis canis (acc. no. AY321119) (n = 2), deposited in the GenBank database. The partial 18S rDNA sequences of Babesia EUI and B. c. canis obtained by us from I. ricinus have been deposited in GenBank, accession nos. GQ325619 and GQ325620, respectively. The results obtained suggest the possible role of I. ricinus as a source of microorganisms, which have been identified as agents of human and canine babesiosis, respectively, in Europe. To our knowledge this is the first report on the occurrence of Babesia EUI and B. c. canis in I. ricinus in Poland.

The purposes of this study were to specify the occurrence and prevalence of Babesia canis, Borrelia burgdorferi sensu lato, and Anaplasma phagocytophilum in ticks removed from dogs in Warsaw, and to determine the Borrelia species occurring in Ixodes ricinus ticks. Among 590 collected ticks, 209 were identified as I. ricinus, and 381 as Dermacentor reticulatus. DNA of B. canis was detected in 11% of D. reticulatus ticks. We found that 6.2% of I. ricinus ticks harbored B. burgdorferi s.l. specific DNA and 2.9% harbored A. phagocytophilum DNA. In these samples sequencing of the detected Borrelia amplicon confirmed infection with Borrelia afzelii genospecies. New sequences were submitted to the GenBank database (accession no. EU152128, EU152127, EU152126). This work is the first detection of B. afzelii and A. phagocytophilum in ticks from Warsaw, and the first survey for the prevalence of B. canis, B. afzelii, and A. phagocytophilum in ticks in central Poland.

Babesia canis has generally been considered the only large Babesia to infect dogs. In this study, we used PCR to detect and characterize B. canis canis isolated from naturally infected dogs in Poland by amplifying and sequencing a portion of the 18S ribosomal RNA (rRNA) gene. Venous blood samples were collected from 76 Babesia-symptomatic dogs. A 559-bp fragment of the B. canis canis 18S rRNA gene was amplified by PCR. The PCR products were then digested with HincII restriction enzyme, and isolates were classified according to whether they were cut (group A) or not (group B) by this endonuclease. Sequencing of the PCR products from the isolates led to the identification of seven sequence variants (four in group A, and three in group B). Sequences were compared with GenBank sequences, and alignments showed that all B. canis canis isolates from Europe may be classified into groups A or B as defined in our study.

In the present study, PCR has been applied to detect and analyze DNA of Babesia spp. extracted from Ixodes ricinus ticks. Collection of I. ricinus was made in 6 forested areas of Zachodniopomorskie Voivodship, Poland, during 2 seasonal peaks of tick activity, i.e., spring and autumn, 2001. In total, 1,328 I. ricinus were collected and processed for PCR with F34 and R323 primers. Babesia spp. was detected in 28 (2% of 1,328 tested) ticks; 26 were identified as B. divergens. The other 2 were identified as B. microti. PCR was conducted with 18S rRNA specific primers and sequencing was processed to precisely identify and compare these isolates with B. microti and B. divergens sequences from Europe, North America, and Asia obtained from the GenBank. Analysis revealed that sequences of B. microti from northwestern Poland are almost identical (99.94%) with those referred to as "Munich strain"; both form a clade different from other European strains, as well as those from Asia and North America (called B. microti, sensu stricto). An investigation performed with B. divergens sequences showed that the sequence from northwestern Poland is 99.94% homologous to an isolate from Ireland ("Purnel"), and differs in just a few nucleotides from other European sequences. Phylogenetic analysis revealed that the sequence of B divergens isolated from Polish ticks form a group that comprise 4 European sequences from Great Britain and Ireland and is, therefore, closely related to other European and North American B. divergnens sequences.

The aim of the study was to establish the role of forest birds as reservoirs of Anaplasma phagocytophilum and Babesia spp. in Wielkopolski National Park. A total of 108 birds from 9 species were collected between May-September 2002. Blood samples were taken from 84 specimens and 442 individuals of the common tick, Ixodes ricinus, were collected from the birds. The 73 additional ticks were collected from vegetation. PCR amplification of a fragment of the epank 1 gene and 18S rRNA gene was used for detection of A. phagocytophilum and Babesia spp. DNA, respectively. Pathogen DNA was not detected in any of the blood samples or ticks collected from birds. On the other hand, 3 ticks collected from vegetation (4.1% of all examined specimens) were positive for A. phagocytophilum DNA. In spite of the high level of infestation of birds by I. ricinus, it is clear that they do not constitute a competent reservoir of A. phagocytophilum and Babesia in WNP. Additionally, I. ricinus is not a significant vector in this area.

The species of genus Babesia and Thelieria are obligate intracellular pathogens that multiply in both vertebrate and invertebrate hosts. Some species of Babesia cause bovine babesiosis infecting erythrocytes of the cattle and wild ruminants, and undergo a complex developmental cycle in ticks which serve as biological vectors. Majority of Theileria spp. cause bovine theileriosis infecting lymphocytes as well as erythrocytes of the cattle and wild ruminants, and similar to Babesia undergo a complex developmental cycle in ticks. In this study, hunter killed roe deer (Capreolus capreolus) and red deer (Cervus elaphus) from north-western Poland were tested for Babesia and Theileria infection in two seasons (spring and autumn, 2004). Infection with babesias and theilerias was detected by PCR assay based on the fragment of nuclear small subunit rRNA gene (nss-ribosomal DNA). Four types of products different in size were obtained and then sequenced. Sequence analysis of nucleotides showed that two kinds of products (385 and 475 bp) were unspecific, the third was characteristic for Theileria sp. (430bp) and the last one for Babesia divergens (407bp). We found that 24.4% of the animals examined were infected with Babesia divergens and 11% with Theileria sp. Percentage of infected animals with B. divergens was almost equal in the spring and autumn (24.6 and 24% respectively). Infection with Theileria was lower in the spring than in the autumn (10.5 and 12% respectively).

Infections caused by the spirochete Borrelia burgdorferi sensu lato may be accompanied by other microorganisms, such as Anaplasma, Ehrlichia and Babesia. These pathogens are transmitted by the ticks and are a risk to humans and animals. Ixodes ricinus ticks collected from recreational areas of Szczecin and northwestern Poland contained DNA of the pathogens mentioned above and cases of double and triple coinfection have been documented. The aim of this paper was to determine if dogs suspect to tick infestation in the area of study are a reservoir for these pathogens and to examine the possibility of coinfection. Canine blood was sampled, part of the material originated from dogs exhibiting symptoms of borreliosis. In an earlier study, the samples were screened for DNA from B. burgdorferi sensu lato. In order to screen for A. phagocytophila and Babesia sp. DNA, a PCR-based method was used with the following primers: EHR521/EHR747 for Anaplasma and FOR1/REV1 for Babesia. In 192 samples only two contained A. phagocytophila DNA. One of these samples originated from a healthy canine, the other from an individual with symptoms of borreliosis. The examined samples were not positive for Babesia sp. DNA. Coinfection was not discovered. The low level of A. phagocytophila infection may indicate that the domestic dog is not a reservoir for Anaplasma and Babesia in Szczecin and northwestern Poland. Moreover, this area does not have populations of the brown dog tick (Rhipicephalus sanguineus) or Dermacentor reticulates--both of which are vectors of E. canis and B. canis and commonly induce ehrlichiosis and babesiosis in canines.

The tick Ixodes ricinus may carry microorganisms which cause serious human and animal diseases, i.a., the Lyme disease (borreliosis), caused by the spirochaete Borrelia burgdorferi sensu lato and babesiosis, induced by the protozoan Babesia microti. Both microbe species may co-occur in the same and other species of the genus tick and produce a mixed infection in humans and animals. The major objective of the study was to identify DNA of B. burgdorferi and B. microti in the I. ricinus ticks collected in spring and autumn 1999 from 6 sites in north-western Poland. The microbial DNA was identified with polymerase chain reaction (PCR). The marker used to detect the B. burgdorferi s.l. DNA was a fragment of the fla gene encoding the protein flagellin, while the B. microti DNA was detected with a fragment of the gene encoding 16S rRNA. A total of 550, 1,160, and 385 tick adults, nymphs, and larvae, respectively, were examined. Among the 155 (7.4%) B. burgdorferi- infected ticks and the 130 (6.2%) infected with B. microti, mixed infection was detected in 0.6% of individuals. The prevalence of coinfection differed between the tick developmental stages. Coinfection was most prevalent (3.1%) in females, males and nymphs being less affected (0.4 and 0.2%, respectively). No coinfection was revealed in the tick larvae. The study described was the first of its kind to be conducted in the former District of Szczecin. For the phenomenon of microbial co-occurrence and related mixed infections to be properly evaluated, the research will be continued.

Babesia microti and B. divergens, the etiological agents of human babesiosis, are transmitted by the bite of Ixodes ricinus. The purpose of this study was differentiation of those two species in ticks collected in urban woods in the city Szczecin (north-western Poland). The prevalence of the DNA of Babesia were investigated by PCR amplification with primers to the fragment from a gene encoding the nuclear small-subunit ribosomal RNA (SS-rDNA). We examined a total of 533 specimens of Ixodes ricinus. The mean infection rate was 16.3%. Our results indicate that a B. microti and B. divergens--specific PCR test may provide a sensitive tool also for the laboratory diagnosis of human babesiosis.

Human babesiosis is caused predominantly by B. microti and B. divergens, a protozooan parasites of red blood cells. Both are transmitted by Ixodes ricinus ticks, also the primary vector of Lyme disease. Clinical manifestation varied widely from asymptomatic infection to a serve rapidly fatal disease. The diagnosis of babesiosis include examination of stained blood smers, serological evaluation indirect antibody tests and PCR. With the evolution PCR--based techniques, the diagnosis and monitoring of babesial infections became more sensitive and reliable.