Search

The prepared article by the team of authors aims to show research in the field of strengthening and developing knowledge and awareness from environmental education in the community of nature conservation services and the academic community. This paper is the result of research conducted in 2014–2015, in the Włoszczowa-Jędrzejów Protected Landscape Area, in the Świętokrzyskie Province. The material for the study was acacia robinia (Robinia pseudoacacia L.) leaves collected on two research areas, differentiated due to the influence of anthropogenic factors. As a result of the study, 5,000 black locust leaves were collected, 65% of which were found to be damaged. Research stands under the influence of strong anthropopressure were characterised by a higher number of lesions on leaves.

Tick-borne encephalitis virus (TBEV) is the most important tick-transmitted arbovirus causing human disease in Europe, but information on its endemic occurrence varies between countries because of differences in surveillance systems. Objective data are necessary to ascertain the disease risk for vaccination recommendations and other public health interventions. In two independent, separately planned projects, we used real-time RT-PCR to detect TBE virus in questing ticks. In Poland, 32 sampling sites were selected in 10 administrative districts located in regions where sporadic TBE cases were reported. In Germany, 18 sampling sites were selected in two districts located in a region with high TBE incidence. Altogether, >16 000 ticks were tested by real-time RT-PCR, with no sample testing positive for TBEV. A systematic search for published studies on TBEV prevalence in ticks in Poland and Germany also suggested that testing large numbers of collected ticks could not consistently assure virus detection in known endemic foci. Although assignment of results to administrative regions is essential for TBE risk mapping, this was possible in only 10 (investigating 22 417 ticks) of 15 published studies (>50 000 ticks) identified. We conclude that the collection and screening of ticks by real-time RT-PCR cannot be recommended for assessment of human TBE risk. Alternative methods of environmental TBEV monitoring should be considered, such as serological monitoring of rodents or other wildlife. [ABSTRACT FROM AUTHOR]

In Europe, Ixodes ricinus tick is the vector of Lyme disease spirochetes and their relatives ( Borreliella genus) and Borrelia miyamotoi . However, a newly described tick I. inopinatus with similar biological features and separated from I. ricinus may act as a vector for different Borrelia species. To date, eleven Borreliella species were detected in the natural populations of I. ricinus . Recently, two North American species have been detected in ticks parasitizing bats and red foxes in Europe, i.e., B. lanei and B. californiensis pointing to the necessity for searching for them in natural tick populations. In this study, using the coxI molecular marker only I. ricinus was identified in field-collected ticks with the exception of individual specimens of Haemaphysalis concinna . Using the flaB gene and mag - trnI intergenic spacer as molecular markers 14 Borreliaceae species have been detected with various frequencies in different parts of northern Poland. Among infected ticks, the most frequent were Borreliella ( Bl. ) afzelii (29.4%) and Bl. garinii (20.0%), followed by Bl. spielmanii , Bl. valaisiana , Bl. lanei , Bl. californiensis , B. miyamotoi , Bl. burgdorferi , Bl. carolinensis , Bl. americana , B. turcica , Bl. lusitaniae , Bl. bissettiae and Bl. finlandensis . Three of the above-mentioned species, i.e., Bl. lanei , Bl. californiensis and B. turcica were detected in this study for the first time in the natural ixodid tick population in Europe. The existence of the newly detected spirochetes increases their total diversity in Europe and points to the necessity of careful identification and establishment of the actual distribution of all Borreliaceae species transmitted by I. ricinus .

One of the most dangerous pests of cereals is Zabrus tenebrioides and, in Poland, it is becoming a serious pest. Entomopathogenic nematodes (EPNs) seem to be a very promising, biological control agent for this pest. Native EPN populations are well adapted to local environmental conditions. The current study characterized three Polish isolates of the EPN Steinernema feltiae , which differed in their effectiveness against Z. tenebrioides . In the field, isolate iso1Lon reduced the pest population by 37%, compared with 30% by isolate iso1Dan and 0% by the iso1Obl isolate; the number of plants damaged by Z. tenebrioides in the presence of the different isolates reflected the results in terms of the decrease in pest population size. After incubation in the soil for 60 days, recovered EPN juveniles of all three isolates were able to infect 93-100% of the test insects, with isolate iso1Obl again showing the lowest effectiveness. The juveniles of isolate iso1Obl were also morphometrically distinct from the other two isolates, as revealed by principal component analysis (PCA), which helped to distinguish the EPN isolates. These findings showed the value of using locally adapted isolates of EPNs; two of the three isolates randomly selected from Polish soil outperformed a commercial population of S. feltiae .

The role of red fox, Vulpes vulpes , and its associated ticks in maintaining Borrelia burgdorferi sensu lato (s.l.) was studied. A total of 1583 ticks were removed from ears of 120 infested animals and were identified as species using a nested PCR targeting the ITS2 and coxI fragments of Ixodes DNA. Ixodes kaiseri prevailed (76%), followed by I. canisuga , I. ricinus , and I. hexagonus . In total, 32.4% of 943 ticks revealed Borrelia DNA and 10 species of B. burgdorferi s.l. complex were identified. Borrelia garinii and B. afzelii comprised 70% of all infections. The other eight species included B. americana, B. bissettiae , B. burgdorferi sensu stricto (s.s.), B. californiensis, B. carolinensis , B. lanei , B. spielmanii , and B. valaisiana . Analysis of tissues from 243 foxes showed that 23.5% were infected with B. burgdorferi s.l. Borrelia garinii was detected in 91% of the infected animals, including 31% of mixed infections with B. afzelii , the second most prevalent species, followed by B. spielmanii . The predominance of B. garinii in PCR-positive animals and infected larval ticks (38.1%), suggests that this spirochete and B. afzelii are preferentially associated with foxes. Although red foxes are exposed to a high diversity of B. burgdorferi s.l. species found in engorged Ixodes ticks, their reservoir competence for most of them appears to be low.

Bats comprise one quarter of the world's mammal species. In Europe, three nidicolous Ixodes tick species, I. vespertilionis, I. simplex and I. ariadnae are specifically associated with cave-dwelling bats, but their role as potential vectors of zoonotic agents is unknown. In this study, we used PCR-based methods to provide the first evidence of Borrelia burgdorferi sensu lato (s.l.) infections in the three bat-associated tick species collected from ten bat species sampled in Poland and Romania. B. burgdorferi s.l. was detected in 24% (64/266) of tick samples, and 40.3% (60/149) of the bats carried infected chiropterophilic ticks. In Poland, the B. burgdorferi s.l. infection prevelance of I. ariadnae ticks parasitizing Myotis species was four times higher compared to the I. vespertilionis ticks derived from Rhinolophus hipposideros bats (44.4% vs.10%, respectively). The observed differences in infection prevalence could be explained by differences in reservoir potential between bat species. Bats from the genus Myotis and Miniopterus schreibersii carried more infected ticks than R. hipposideros regardless of the tick species. Analysis of the flaB gene sequences revealed seven species from the B. burgdorferi s.l. complex (B. afzelii, B. carolinensis, B. garinii, B. lanei, B. spielmanii, B. burgdorferi s.s., and B. valaisiana), of which five are considered as human pathogens. This large diversity of Borrelia species may reflect differences in susceptibility of chiropteran hosts and/or the tick vectors. Generally, mammal-associated B. burgdorferi s.l. species were more common than bird-associated species. Our study provides evidence for new enzootic transmission cycles of B. burgdorferi s.l. spirochetes involving nidicolous Ixodes tick species and cave-dwelling bats., (Copyright © 2019 Elsevier GmbH. All rights reserved.)

European badgers and raccoon dogs and their associated ticks and lice were assayed for the presence of Lyme borreliosis and relapsing fever-group spirochete DNA in western Poland. Analyses of blood, ear-biopsy and liver samples revealed that 25% of 28 raccoon dogs and 12% of 34 badgers were PCR positive for borreliae. Borrelia garinii was the dominant species in raccoon dogs (62.5%), followed by B. afzelii (25%) and B. valaisiana (12.5%). PCR-positive badgers were infected only with B. afzelii. A total of 351 attached ticks was recovered from 23 (82%) of the raccoon dogs and 13 (38%) of the badgers. Using a nested PCR targeting the ITS2 fragments of Ixodes DNA, four Ixodes species were identified: I. ricinus, I. canisuga, I. hexagonus, and one provisionally named I. cf. kaiseri. Ixodes canisuga and I. ricinus prevailed on both host species. The highest infection prevalence was detected in I. ricinus, followed by I. canisuga and I. cf. kaiseri. Borrelia garinii and B. afzelii accounted for 61.6% and 30.1% of the infections detected in all PCR-positive ticks, respectively. Four other Borrelia species (B. burgdorferi sensu stricto, B. valaisiana, B. lusitaniae and B. miyamotoi) were detected only in I. ricinus from raccoon dogs. Moreover, Borrelia DNA, mostly B. garinii, was detected in 57 (81.4%) of 70 Trichodectes melis lice derived from 12 badgers. The detection of B. afzelii in one-half of PCR-positive biopsies reconfirms previous associations of this species with mammalian hosts, whereas the high prevalence of B. garinii in feeding lice and I. ricinus ticks (including larvae) demonstrates that both carnivores serve as hosts for B. garinii. The lack of B. garinii DNA in the tissues of badgers versus its prevalence in raccoon-dog biopsies, however, incriminates only the latter carnivore as a potential reservoir host., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

Ixodes ricinus has the potential to transmit zoonotic pathogens to humans and domestic animals. The feeding I. ricinus (n = 1737) collected from 49 Shetland ponies and questing ones from vegetation (n = 371) were tested for the presence and differentiation of the bacterial species. DNA of I. ricinus ticks was examined with PCR and sequencing analysis to identify species of Borrelia burgdorferi sensu lato (Bbsl), Anaplasma phagocytophilum and Rickettsia spp. Altogether, 24.3 % I. ricinus of the infested horses and 12.4 % ticks from vegetation carried at least one pathogen species. Horse-feeding ticks (19.2 %) were significantly more frequently infected with Borrelia spp. than questing ticks (4.8 %). Among Bbsl species, in I. ricinus infesting ponies, B. garinii, B. afzelii, B. burgdorferi sensu stricto, B. valaisiana and B. lusitanie and one species, B. miyamotoi related to relapsing fever group, were detected. The 73 flaB gene sequences of Borrelia obtained from feeding I. ricinus have been deposited in GenBank. Among Rickettsia species, two were identified: R. helvetica which was dominant and R. monacensis. Infections with more than one pathogenic species, involving mostly Bbsl and R. helvetica were detected in 6.3 % of infected ticks collected from horses. Shetland ponies may play an important role in the epidemiological cycle of Bbsl and probably could contribute to the natural cycle of A. phagocytophilum and R. helvetica as host for infected ticks. The awareness about these infectious agents in ticks from ponies might be an important criterion for the risk assessment of human diseases, especially as these animals are maintained for recreational purposes.

Forest animals play fundamental roles in the maintenance of Ixodes ricinus and Borrelia species in the forest biotope. To identify the forest vertebrate species that are host for I. ricinus and for the recognition of the reservoirs of Borrelia species, the blood-meal of 325 I. ricinus ticks collected at two forest sites in north-western Poland were analysed. Nested PCR was used to detect polymorphisms in a fragment of the mitochondrial 12S rRNA gene for the identification of the hosts species. The products were digested with the restriction enzymes, a combination that allows the identification of 60 vertebrate species, comprising 17 bird, 4 reptile and 39 mammalian species. Host DNA was detected in 244 (75%) I. ricinus individuals, with the species being detected and classified for 210 (86%) samples. The restriction patterns resulted in the identification of 14 vertebrate species, including 2 species of birds, lizard, badger, rabbit, deer; most of the samples contained DNA from wild boar (Sus scrofa), red fox (Vulpes vulpes), red deer (Cervus elaphus) and roe deer (Capreolus capreolus). Identification of Borrelia species was based on the flaB gene using nested PCR coupled to RFLP. This method allows the identification of all Borrelia species transmitted by I. ricinus in Europe, including B. miyamotoi and 3 genetic variants of B. garinii. In the studied isolates, 2 species belonging to B. burgdorferi sensu lato were identified--B. garinii and B. afzelii, and B. miyamotoi, which are related to relapsing fever borreliae.

Background: Borrelia burgdorferi sensu lato includes at least 20 species in the world, and half of these are found in Europe. The usefulness of high resolution melting (HRM) analysis of DNA denaturation curves has been assessed for differentiation of Borrelia species., Methods: HRM protocol for Borrelia species was used to examine the 77 DNA extracts selected from earlier studies with the use of three different molecular markers: flaB, rplL, and groEL., Results: The studies revealed that the best marker is the groEL gene, which enables identification of 8 Borrelia species, including B. miyamotoi from the relapsing fever borreliae group and 7 of B. burgdorferi s.l. complex (B. garinii, B, afzelii, B. burgdorferi s.s., B. valaisiana, B. lusitaniae, B. bissetii, B. spielmanii)., Conclusions: The HRM method, when compared with other PCR variants with regard to the reduced time of analysis, is an alternative for the procedures used in the molecular diagnostics of borreliosis including testing of blood samples or saved Ixodes ticks for the presence and genotyping of Borrelia burgdorferi after biting a patient.

DNA analysis of blood meals from unfed nymphal Ixodes ricinus allows for the identification of tick host and tick-borne pathogens in the host species. The recognition of host species for tick larvae and the reservoirs of Borrelia, Rickettsia and Anaplasma species were simultaneously carried out by analysis of the blood meals of 880 questing nymphal I. ricinus ticks collected in forest parks of Szczecin city and rural forests in northwestern Poland that are endemic areas for Lyme borreliosis. The results obtained from the study indicate that I. ricinus larvae feed not only on small or medium animals but also on large animals and they (i.e. roe deer, red deer and wild boars) were the most prevalent in all study areas as the essential hosts for larvae of I. ricinus. The composition of medium and small vertebrates (carnivores, rodents, birds and lizards) provided a more diverse picture depending on study site. The reservoir species that contain the most pathogens are the European roe deer Capreolus capreolus, in which two species of Rickettsia and two species of Borrelia were identified, and Sus scrofa, in which one Rickettsia and three Borrelia species were identified. Rickettsia helvetica was the most common pathogen detected, and other included species were the B. burgdorferi s.l. group and B. miyamotoi related to relapsing fever group. Our results confirmed a general association of B. garinii with birds but also suggested that such associations may be less common in the transmission cycle in natural habitats than what was thought previously.

This study examined the possibility of applying a new diagnostic method, high resolution analysis of DNA denaturation curve (high resolution melting - HRM), for identification of Borrelia species. DNA samples were obtained from Ixodes ricinus ticks collected from vegetation and removed from hunted roe deer. For differentiation of Borrelia species, the HRM protocol based on the analysis of the groEL gene was applied. A product characteristic for Borrelia was obtained in 19/123 samples (15.4%). The studied isolates were classified as four species: B. garinii, B. valaisiana, B. afzelii and B. miyamotoi. Two separate groups of isolates within the B. afzelii species were also found. The results show that the groEL gene is useful for rapid differentiation of B. burgdorferi sensu lato with the HRM method from different extracts of DNA and it also allows precise differentiation of Borrelia species and strains. The HRM method shortened and simplified detection and differentiation of Borrelia species from different biological sources.

A new protocol employing nested PCR-restriction fragment length polymorphism (RFLP) based on the flaB gene and two restriction enzymes was worked out. This protocol allows the identification of all Borrelia species transmitted by Ixodes ricinus in Europe, including Borrelia miyamotoi and 3 genetic variants of B. garinii. A dendrogram of flaB sequence similarity was in accordance with RFLP variants.

Borrelia burgdorferi sensu lato, carried by Ixodes ticks, is one of the most significant human pathogens, causing Lyme disease. As there is no standardized PCR method for detection and identification of spirochaete DNA, we carried out a comparative analysis using a set of complementary primers for three regions in the genomic DNA of these bacteria (genes fla and rrs and the non-coding rrs-rrlA region). DNA extracted from 579 Ixodes ricinus ticks was subjected to nested PCR. DNA of the examined spirochaetes was detected in 43 (7.4 %) lysates when the fla gene was used as a molecular marker, in 7 (1.2 %) lysates when using primers complementary to the rrs gene, and in 12 (2.1 %) lysates using primers complementary to the non-coding rrs-rrlA sequence. RFLP analysis based on the fla gene helped identify species from the B. burgdorferi sensu lato complex (B. burgdorferi sensu stricto, Borrelia afzelii, Borrelia garinii, Borrelia valaisiana), detect co-infections, and also identify Borrelia miyamotoi. Therefore, the fla gene is the most sensitive and specific molecular marker for the detection and identification of Borrelia spirochaetes in I. ricinus.

In the wake of controversies surrounding the usefulness of PCR in the diagnostics of borreliosis, the aim of the presented study was to monitor the presence of B. burgdorferi s.l. in dogs with clinical borreliosis in the course of relevant treatment. The monitoring was based on detecting borrelia's DNA before- (study I), during- (study II), and after completion of the therapy (study III). In addition, to rule out possible coinfections, the dogs' blood was examined for the presence of anaplasma, babesia and rickettsia. Blood samples taken from 11 dogs, with clinically detected borreliosis, were used for obtaining DNA for PCR. Positive results of PCR, with primers complementary to the fla gene, indicating the presence of DNA of B. burgdorferi s.l., were noted, in study I, in the blood of 7 dogs (63.6% dogs), in study II in 3 dogs, while in study III all blood samples were negative. In 6 out of 7 PCR+, the first study was carried out during week 1. Therefore, the PCR method is useful for monitoring early canine infections with spirochetes B. burgdorferi s.l. In all positive samples, subjected to PCR-RFLP, it was the case of a single genospecies, i.e. B. burgdorferi sensu stricto. Studies for the presence of DNA of Babesia sp., as well as DNA of Ricettsia helvetica, were negative in all samples. Anaplasma phagocytophilum DNA was detected in the blood of a single dog, and only in study I. The same dog also proved positive for the presence of borrelia DNA. Co-occurrence of both pathogens did not disturb the clinical picture of borreliosis, and the administered treatment was also effective for the mixed infection.

In this study we present the nested RT-PCR strategy designed for detection of TBEV RNA in ticks Ixodes ricinus. The presented nested RT-PCR method using 2 different primer pairs specific primers for NS5 gene provides specific TBEV cDNA detectable by electroforesis in agarose gel. Of the 177 polls of ticks investigated, TBEV RNA was detected in 14, which accounts for 7.9% of all pools. We confront the PCR results of tested ticks to routine surveillance data. The obtained results showed that the TBEV RNA is detectable in ticks collected in areas in Poland, which are defined as an non-endemic. The nested RT-PCR method can be used as a tool of epidemiological surveillance as well as for screening of occurrence of circulating TBEV.

Borrelia burgdorferi sensu lato (s.l.) complex, the etiological factor of Lyme disease, includes over a dozen species of bacteria and 3 pathogenic within it. According to many authors, the clinical symptoms of borreliosis depend on the species that cause the disease. The most frequent symptom of early localized borreliosis is erythema migrans (EM). The aim of the research was to determine species of B. burgdorferi s.l. in 32 patients from the Western Pomerania region in whom EM has been recognized. Blood samples of patients were investigated by PCR-RFLP method, with the use of enzyme differentiating species. The DNA of spirochetes was detected in 25 patients (25/32, 78.1%), compared with 23/32 (71.8%) of ELISA positive patients. Among 25 positive samples, 10 contained the DNA of B. garinii (10/25, 40%), 5 the DNA of B. afzelii (5/25, 20%), 4 the DNA of B. burgdorferi sensu stricto (s.s.) (4/25, 16%) and in 6 samples (6/25, 24%) the DNA of both B. garinii and B. afzelii was found. The DNA of B. burgdorferi s.l. spirochetes may be detected in patients with EM after antibiotic treatment. The most frequent species in patients with EM from the Western Pomerania region is B. garinii. Infections with more than one species of B. burgdorferi s.l. may occur in patients with EM.

Background: Red deer (Cervus elaphus) is one of the most important host of the adult tick (Ixodes ricinus) which is the basic vector of the Lyme disease causative agent--Borrelia burgdorferi sensu lato in Europe. The aim of the present study was to establish the role of red deer in the transmission of B. burgdorferi s.1. Material and methods. Tissues from 74 red deers were evaluated and the presence of B. burgdorferi s.1 DNA was identified using nested PCR technique based on fla gene. The identification of species belonging to B. burgdorferi s.1 complex was performed after restriction digestion of nested PCR product with Ddel enzyme and sequencing of nested PCR product. The study included also 55 isolates of I. ricinus females removed from red deer and 466 ticks (73 adult and 393 nymphs) collected from the vegetation in the area where the red deer lives., Results: There were no DNA of B. burgdorferi s.1 complex in the red deer tissues and in ticks removed from deer, however in one tick removed from deer the DNA of other Borrelia species--B. miyamotoi was identified. In ticks collected from vegetation 3 species belonging to B. burgdorferi s.1. complex were identified: B. garinii (3.2% ticks studied), B. afzelii (6.9%) and B. valaisiana (3.6%), however DNA of B. miyamotoi was absent. These results confirm inability of survival of B. burgdorferi s.1. species in tick I. ricinus feeding on red deer blood. However there is a possibility of survival of B. miyamotoi in presence of deer blood at least in ticks feeding on red deer. The main role of red deer in keeping the constant infection level of B. burgdorferi s.1. in the whole population of I. ricinus ticks does not concern B. miyamotoi.

The aim of the study was to assess the frequency of Borrelia burgdorferi DNA detection in the blood and urine of patients diagnosed with erythema migrans, and compare the results of PCR-based methods with ELISA methodology. The latter was used to detect serum antibodies against Borrelia burgdorferi of the IgM and IgG classes, before and after antibiotic therapy. The study included 86 patients hospitalized in the Department of Infectious Diseases and Neuroinfections in the Medical Academy in Białystok, diagnosed with the erythema migrans phase of Lyme borreliosis. Examinations were carried out twice: the first at the moment of diagnosis (Trial 1), the second after 4 weeks of antibiotic therapy. The study showed that antibiotic therapy in the early phase of borreliosis does not decrease the sensitivity of PCR and that after 4 weeks of therapy (Trial 2), spirochete DNA is still detectable in most patients (45/86). There was no correlation between detectability of spirochete DNA and the presence of antibodies against B. burgdorferi s.l. (assessed by ELISA) during the course of erythema migrans. The largest percentage of positive results in the detection of B. burgdorferi s.l. DNA was observed in patients who simultaneously possessed IgM and IgG antibodies against B. burgdorferi, while the lowest percentage of PCR positive results was among patients with only IgM antibodies.

Forest passerine birds and their ectoparasites: Ixodes ricinus ticks and Syringophilidae quill mites were surveyed for infection with Anaplasma phagocytophilum in west-central Poland. Of 126 birds captured from May to June of 2002, 71 (56.3%) comprising eight species, hosted immature I. ricinus ticks. A total of 383 ticks and 71 blood samples collected from tick-infested birds were investigated by PCR. The pathogen was not detected in either bird-derived ticks or in blood samples. Among the captured birds, a total of 14 individuals representing four species hosted quill mites from the family Syringophilidae. Three of the 14 mite pools recovered from the 14 mite-infested birds harbored A. phagocytophilum DNA by amplifying both the epank1 and p44 gene. The PCR-positive pools originated from one blackbird and two starlings. The specific biology of syringophilid mites, which parasitize exclusively inside the quill of feathers, feeding on host subcutaneous fluids, implies that they must have acquired the pathogen from a bacteremic bird. These results provide the first indirect evidence that at least some passerine hosts are prone to develop systemic infection with A. phagocytophilum under natural conditions. Consequently, the infected quill mites may serve as a "biological marker" of past or current infection with the agent within birds.

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.

Wild rodents and the subadult Ixodes ricinus (L.) ticks infesting them were examined for the presence of Borrelia burgdorferi Johnson, Schmid, Hyde, Steigerwalt & Brenner s.l. in a sylvatic habitat in west central Poland during May-September 2002. In total, 818 feeding ticks were recovered from 73 infested yellow-necked mice, Apodemus flavicollis Melchior; in addition, bank voles, Clethrionomys glareolus Schreber, were rarely captured and proved to be weakly parasitized. Only 2.7% of A. flavicollis and 2.2% of 320 engorging larvae were polymerase chain reaction (PCR) positive for the bacterium. All spirochete-PCR-positive samples yielded exclusively B. burgdorferi s.s. This genospecies was also the most prevalent in questing nymphs and accounted for 87.5% of the total number of Borrelia infections in nymphal ticks collected during May and June 2 yr later. The presence of the same genospecies both in naturally engorged larvae and blood-positive animals as well as the high predominance of B. burgdorferi s.s. in questing nymphs strongly differs from most study sites investigated in Europe. This unique pattern of Borrelia-diversity in both rodents and ticks seems to be determined by highly site-specific host vertebrate cenosis, and yellow-necked mice are involved in the maintenance of B. burgdorferi s.s. in the forest habitat. However, the transmission efficiency of this spirochete from the mice to the I. ricinus vector seems to be very low. The research provides additional information on the complexity of B. burgdorferi s.l. ecology in Europe, pointing to the importance of the local host community.

European genospecies of B. burgdorferi sensu lato were identified by examining unfed I. ricinus ticks collected from 10 locations in northwest Poland. Research was conducted using 3 methods: PCR amplification of the fla gene with the FLA1 and FLA2 primer set conserved in all European species of B. burgdorferi sensu lato, PCR-RFLP, and sequencing. There were 5 restriction patterns obtained in this study: 4 characteristic for genospecies B. burgdorferi sensu stricto, B. garinii, B. valaisiana, and B. afzelii and 1 untypical restriction pattern type. PCR products for all restriction patterns were sequenced. Polish sequences of the fla gene for B. burgdorferi s.s., B. garinii, B. valaisiana, and B. afzelii were identical with sequences from GeneBank at 99.79%, 99.58%, 100% and 100% respectively. The fifth sequence demonstrated 99.79% identity with sequences of a B. lusitaniae PotiB2 isolate from Portugal, and also clusters with this strain. B. lusitaniae DNA in I. ricinus was detected in 3 out of 10 localities and constituted 5.9% of infected individuals with I. ricinus.

The aim of the paper was an attempt to correlate clinical signs with the presence of DNA of Borrelia burgdorferi (sensu lato) s.l. and the antibodies against B. burgdorferi s.l. in the blood of dogs. Among the animals studied there were 62 dogs delivered to the Veterinary Clinic in Szczecin and 30 from the Municipal Animal Shelter in Szczecin with varied clinical signs of borreliosis. In all cases the owners admitted frequent contacts of their dogs with ticks, both in the past, as well as shortly before the onset of sickness. We used two methods: PCR for detecting DNA of B. burgdorferi s.l. and ELISA test for detecting antibodies against the spirochete. Lameness, the principal symptom of canine borreliosis was the most frequent symptom of the group of 31 PCR-positive animals. The other most common symptoms in PCR-positive dogs were fever, swelling of joints and loss of body weight. DNA of B. burgdorferi s.l. was most frequently detected in the blood of dogs of the group 2-5 years old (13/54.1 %). ELISA tests specific for IgG antibodies were positive in 37 of 92 sera (40.2 %) taken from examined dogs. Lameness was observed in 15 of 37 IgG-seropositive dogs and in 25 of 55 seronegative animals. In 54 % of dogs with the antibodies, swelling of instep- and wrist joints was observed compared to only 24.4 % in seronegative dogs. An attempt to correlate the PCR results with the results of tests detecting antibodies against B. burgdorferi s.l. revealed that fewer than half (45.1 %) of the dogs with presence of DNA of the spirochete, developed an immune response. Therefore the transfer of B. burgdorferi s.l. form, the primary lesion to the target tissues, is possible in dogs which did not develop immune response or develop an insufficient response. Among 92 borreliosis-suspected dogs 54 (over 58 %) were diagnosed positively using laboratory methods. In most cases there was a correlation between clinical symptoms of borreliosis and presence of DNA B. burgdorferi, thus PCR may contribute to improving to a large extent diagnostic of canine Lyme disease.

Canine Lyme borreliosis may be caused by three Borrelia burgdorferi sensu lato genospecies. The prevalence of infection by Borrelia species was determined by nested polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) with the enzyme Fsp4H I in the blood of dogs naturally infested by ticks in an endemic region of Poland. Blood samples were collected from 98 dogs of various breeds, delivered to the Veterinary Clinic in Szczecin (northwestern Poland) for various reasons. Nested PCR revealed the presence of DNA characteristic of only 1 genospecies, i.e. B. burgdorferi sensu stricto (s.s.), in all PCR-positive samples. Digestion of PCR products from a fragment of the fla gene amplified with primers FLA1 and FLA2 gave only one band pattern consistent with the pattern obtained from sequence analysis of the fla gene from a reference isolate of B. burgdorferi s.s. GeHo (X15660) from GenBank.

Unlabelled: The purpose of this work was to evaluate Borrelia burgdorferi presence in peripheral blood of patients with Lyme disease using PCR method. The study was conducted on 96 patients divided into 4 groups depending on clinical form of the disease., Material and Methods: There were 9 patients with erythema migrans, 11 with neuroborreliosis, 36 with Lyme arthritis, 18 with disseminated form of borreliosis and 22 patients whose initial suspicion of Lyme disease was excluded. The diagnosis was based on clinical examination and results of serologic test performed using ELISA method showing serum presence of antibodies anti-borrelia IgM and IgG., Results: The results of PCR examination were analysed depending on clinical form of the disease and result of serologic test. The highest rate of positive results in PCR examination was stated in group of patients showing serum presence of IgM anti-borrelia antibodies (34.48%) and in patients with diagnosed Lyme arthritis (38.88%). The lowest rate of positive PCR results (16.66%) was stated in group of patients with negative serologic test., Conclusions: The probability of Borrelia burgdorferi DNA blood presence was highest (76.0%) in patients with positive serologic test IgM anti-borrelia antibodies and diagnosed Lyme arthritis.

In order to learn the heterogeneity of the DNA of B. burgdorferi s.l. and the prevalence of co-infections of B. burgdorferi s.l. genospecies in the populations of I. ricinus, collected in north-western Poland, the nested PCR method was applied, a fragment of the fla gene being used as a marker. Basing on the prevalence data of B. burgdorferi s.l. DNA in I. ricinus ticks in 8 sampling sites during 1998-2001, it may be stated that a risk of contracting Lyme disease exists in forested areas of north-western Poland, the highest in relation to B. burgdorferi s.s. (76.3% infected ticks), lower by B. garinii (2% infected ticks), and minimal threat being posed by B. afzelii (0.3%). I. ricinus ticks collected in north-western Poland pose a risk of contracting double infection by B. burgdorferi s.l. genospecies, i.e. B. burgdorferi s.s. with B. garinii, and B. burgdorferi s.s. with B. afzelii. The north-western part of Poland represents an endemic area for B. burgdorferi s.l.

The aim of the present study is to establish a common RAPD marker for P. jenningsi using a series of Ro primers and to investigate if strains originating from distant and isolated localities (Japan, China, India, Saudi Arabia) have isolated gene pools and represent distinct species. An analysis of dendrograms constructed on the basis of RAPD-PCR fingerprints with four primers (Ro 460-04, 460-06, 460-07, and 460-10) from the first part of this project (SKOTARCZAK et al. 2004), assigns the strains to two groups consisting of the continental strains (India, Saudi Arabia, China) and Japanese strains that have been considered as a separate sibling species within P. jenningsi. The genetic similarity of the Indian and Arabian strains was ascertained, whereas the Chinese strain formed an independent branch in this sibling species. The primers Ro (460-01,460-02, 460-03, 460-05, 460-08) also distinguish between two groups of strains, although they divide the Japanese strains into two subgroups that are not reproductively isolated. This probably indicates genetic variation within this sibling species. However, it comprises one common gene pool (successful inter-strain crosses) and is reproductively isolated from the other sibling species. The results presented in these papers confirm that the construction of ten band patterns having marker attributes is possible on the basis of DNA amplification from 9 strains of P. jenningsi with the RAPD-PCR fingerprinting method using five primers from the Ro series. The patterns can be assigned to three marker-groups: a general species group, a group differentiating between sibling species, and accessory strain markers.

In order to estimate the risk of contracting Lyme disease in the forest areas of north-western Poland, PCR-based studies were carried out on 6,817 Ixodes ricinus ticks for infection by the spirochaete B. burgdorferi sensu lato (s.l.). The studies were performed using the primers for the fla gene, conserved for all European genospecies of B. burgdorferi s.l. Based on the incidence of B. burgdorferi s.l. DNA in I. ricinus ticks at eight sampling sites during 1998-2001, it may be concluded that a risk of contracting Lyme disease is present in the forest areas of north-western Poland. The highest risk of infection (9.4 % of infected ticks) is posed by human contact with female I. ricinus, and the risk is higher in late spring and early summer than in late summer and early autumn. The north-western part of Poland is an endemic region for B. burgdorferi s.l.

Our earlier studies on ticks, Ixodes ricinus have demonstrated that the north-western part of Poland is an endemic area for Borrelia burgdorferi sensu lato and therefore sick dogs, at the time of the highest activity of ticks, should be suspected for having borreliosis. We carried out a preliminary PCR survey of the blood of 15 dogs naturally exposed on ticks for the presence of the DNA of B. burgdorferi using primers complementary to the fragment of the gene encoding 16S rRNA of the small ribosome subunit. We found 6 out of 15 dogs were infected, although 2 dogs had a lameness - the attribute of canine borreliosis were PCR-negative. Our findings suggest that the exposure to B. burgdorferi is common in dogs in the region declared an endemic area of borreliosis, and that this disease should be important to local veterinarians.

The present studies with application of classical strain crosses and RAPD-PCR analyses showed the existence of different genetic species (syngens) within Paramecium jenningsi. So far the existence of only one syngen has been accepted. It was found that strains from Saudi Arabia, India, and China compose one genetic species (syngen 1) and six strains from Japan compose second genetic species (syngen 2).

Occurrence of borreliosis in human population is associated with possibility of contact with the biological vector of this disease - a common European tick, Ixodes ricinus. Therefore, the highest number of cases of Lyme disease has been recorded among forestry workers and inhabitants of wooded areas. Diagnostics of borreliosis is based on immunoserologic tests - ELISA or indirect immunofluorescence method, Western blot technique, or on increasingly popular DNA examination using the polymerase chain reaction (PCR). In the present study, where 61% of the forestry workers were seropositive, we also tried to find a correlation between the results of serological tests and PCR tests with the clinical state of the patients. Despite finding IgM antibodies in 10 persons tested, which would indicate their recent infection, no DNA of B. burgdorferi s.l. was detected in their blood. Also, no DNA of this bacteria was present in 8 persons with IgM and IgG antibodies. No genetic material of the bacteria was found in persons with IgG antibodies, indicating the possibility of chronic infection. The clinical data suggested past symptomatic infection (ECM), or even more often, asymptomatic infection with B. burgdorferi.

Objective and Methods: Because of lack of standardization of PCR method in Poland to amplify Borrelia burgdorferi s. l. DNA in this case we applied three different primer sets for amplification of: fla gene, 16S rRNA small subunit gene as well as 5S-23S rRNA intergenic spacer and two different polymerases. We compare the efficacy of Borrelia burgdorferi s. l. detection in whole blood from borreliosis suspected patients and isolates of intestine contents of Ixodes ricinus ticks. A quality of polymerase is also important in sensitivity of PCR method., Results: The best sensitivity of PCR was in blood with primers complementary to 16S rRNA gene, then in ticks isolates with primers complementary to fla gene., Conclusion: We affirm that results of PCR detection depend on genetic markers employed to DNA detection and DNA isolation method.

Within the last years, the incidences of diseases transmitted by Ixodes ricinus tick have rapidly increased. We estimated the occurrence of Ixodes ricinus in the popular recreation urban areas in Szczecin and in the Province of Szczecin. The study was carried out in 1999, with two samples at each site, and were compared with data of 1998. The temperature and humidity of air were measured. The most ticks were found in the range of 70-80% relative humidity of air. Among 3.198 specimens collected 59.5% were nymphs, 19.0% larvae, 11.1% females and 10.4% males. The nymphs were the most frequent in spring and in autumn, while the larvae were most frequent in autumn (26.2%) then in spring (11.9%).

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.

Within the last few years, the incidence of Lyme disease has rapidly increased in Europe, with the causative agent of the disease is Borrelia burgdorferi--a spirochete. In Poland, Lyme borreliosis is being identified, mainly, based on the clinical symptoms, epidemiological anamnesis, and serological tests. On the other hand, it is evident from the foreign publications, that in many cases representing different phases of Lyme disease, a reliable and totally accurate identification tool of Borrelia burgdorferi is amplification of bacterial DNA using PCR method. The main goal of the present studies has been implementation of the DNA amplification method into diagnostic procedures of Lyme. Although we dealt with DNA of B. burgdorferi isolated from tics, it would not make a difference because the method of DNA isolation is the same for human samples. The results acquired from the preliminary studies, suggest that amplification of a fragment of fla gene, may be useful in Lyme disease diagnostics. Spirochetes of B. burgdorferi sensu lato were detected in tics Ixodes ricinus using PCR method in both individual animals and tick pools. The latter version of the method seems to be very useful in so called screening studies, because of minimizing the cost and duration of the procedure.

Borrelia burgdorferi sensu lato (s. l.), the etiological agent of Lyme diesease, is transmitted by the bite of Ixodes ricinus. During May and September 1999, field surveys on Lyme disease spirochetes were conducted in three locations of a region of north-west Poland, known as recreational districts visited by many people. The ticks Ixodes ricinus were collected in natural habitats by dragging a flanel cloth over the vegetation. Sex and developmental stage of each tick were determined. Based on a polymerase chain reaction test with primers that recognize a chromosomal gene of all strains, out of the total 1414 specimens collected, 126 (8.9%) were found to be infected. The species B. burgdorferi s. l. comprises at least three pathogenic genomospecies, B. burgdorferi sensu stricto (s. s.), Borrelia garinii, and Borerelia afzelii, witch could be distinguished in nested-PCR tests with species-specific primers. B. burgdorferi s. s. was most prevalent (96% of infected ticks), followed by B. garinii (1.3%), and B. afzelii. was not found. Of the infected ticks, over the 99% were infected with a single species, one specimens was infected with two species. For 4 ticks, the infecting species could not be identied. The difference in rates of prevalence was observed among the tree locations (17%--5.3%--3.2%).

The aim of the study was to estimate the occurrence (the quantative and rate percent) of Ixodes ricinus in the popular recreation areas in Szczecin (Arkonka, Osów, Głebokie, Landscape Park of Szczecin, Dabie Forest Park, Zdroje Forest Park) and in province of Szczecin (Forest of Goleniów, Ińsko, Pobierowo, Chojna). Investigations were performed in 1998 year, twice on each places; in May/June and repeted in September/October. The temperature and humidity of air were measured. Obtained specimens were regard of sex and growing stage during each collection. A total of 2.055 specimens collected 49% were nymphs, 13.9% female, 11.3% male and 25.8% larvaes. The nymphs the most frequently were in spring when humidity of air was 55% and temperature 24 degrees C. The larvaes, in autumn were most frequently (31.4%) then in spring (20.5%) when the temperature of the air was 18-22 degrees C, and the humidity from 60 to 85% during the collections.

Among Polish ticks species the most common Ixodes ricinus has the biggest medical importance. Within the last few years, the incidence of disease transmitted by ticks has rapidly increased. We have made a thorough analysis of the quantative and rate per cent of occurrence of various stages Ixodes ricinus in the forest areas of some places in Szczecin province and in the parks of Szczecin, that are known as highly recreative and frequently visted by many people. A total of 426 (68% numphs) specimens collected there show that ticks frequently occupy habitats closely associated with man.