New problems of environmental ecology: ticks and tick-borne pathogens in city parks of Ukraine.

City parks of Vinnytsia, Ukraine, were examined for the occurrence of host-seeking ticks and collected specimens were examined for presence of rickettsial pathogens. In the summer season, the only collected individuals were Dermacentor reticulatus adults (n = 203). DNA identification of pathogens was conducted with the use of 16S rRNA gene amplification. Molecular analyses confirmed the presence of Rickettsia raoultii in ticks from all parks, with infection prevalence ranging from 5% to 68% across parks. These results indicate that city parks in Vinnytsia have become a new habitat for ticks of high medical and veterinary importance. Due to the risk of acquiring tick-borne pathogens, the urban environment such as parks and home gardens, should be monitored for the presence of ticks and their infection with human pathogens. Screening of these areas is an important preventive element of environmental epidemiology.

The Impact of a Pandemic COVID-19 on the Incidence of Borreliosis in Poland.

PURPOSE: Lyme disease is the most common tick-borne disease, caused by spirochetes of the genus Borrelia, transmitted by ticks of the Ixodes genus in Poland. The purpose of this analysis was whether the COVID-19 outbreak had a significant impact on the number of reported Lyme disease cases. MATERIALS AND METHODS: The data included in the World Health Organization (WHO) and the data from the "Reports on incidence of infectious diseases, infections and poisoning in Poland" presented by the Department of Epidemiology NIZP-PZH were analyzed. RESULTS: To the end of 2020, there were registered 12, 524 Lyme disease cases. In the same period, in 2018 and 2019 were registered, respectively, 20, 150 and 20, 614 Lyme disease cases. The overall number of Lyme disease cases in 2018 and 2019 was at a similar level. The monthly increase in the number of cases was also at a similar level. The year 2020 in January and February was characterized by the same increase in the number of cases as in previous years. The difference started to be noticeable in March and the lowered growth compared to the previous years has been maintained to this day. In December, about 8, 000 fewer cases of Lyme disease were registered than in previous years. CONCLUSION: The reduced number of cases of Lyme disease coincided with the beginning of the COVID-19 epidemic in Poland in March 2020. Every year, the incidence of Lyme disease in Poland is at a similar level with a similar monthly increase. The outbreak of the COVID-19 pandemic had a significant impact on the number of cases recorded, which could have catastrophic consequences for people who did not receive treatment in the right time.

Detection of Tick-Borne Pathogens of the Genera Rickettsia, Anaplasma and Francisella in Ixodes ricinus Ticks in Pomerania (Poland).

Tick-borne pathogens are an important medical and veterinary issue worldwide. Environmental monitoring in relation to not only climate change but also globalization is currently essential. The present study aimed to detect tick-borne pathogens of the genera Anaplasma, Rickettsia and Francisella in Ixodes ricinus ticks collected from the natural environment, i.e., recreational areas and pastures used for livestock grazing. A total of 1619 specimens of I. ricinus were collected, including ticks of all life stages (adults, nymphs and larvae). The study was performed using the PCR technique. Diagnostic gene fragments msp2 for Anaplasma, gltA for Rickettsia and tul4 for Francisella were amplified. No Francisella spp. DNA was detected in I. ricinus. DNA of A. phagocytophilum was detected in 0.54% of ticks and Rickettsia spp. in 3.69%. Nucleotide sequence analysis revealed that only one species of Rickettsia, R. helvetica, was present in the studied tick population. The present results are a part of a large-scale analysis aimed at monitoring the level of tick infestation in Northwest Poland.

Loop-Mediated Isothermal Amplification (LAMP): The Better Sibling of PCR?

In 1998, when the PCR technique was already popular, a Japanese company called Eiken Chemical Co., Ltd. designed a method known as the loop-mediated isothermal amplification of DNA (LAMP). The method can produce up to 109 copies of the amplified DNA within less than an hour. It is also highly specific due to the use of two to three pairs of primers (internal, external, and loop), which recognise up to eight specific locations on the DNA or RNA targets. Furthermore, the Bst DNA polymerase most used in LAMP shows a high strand displacement activity, which eliminates the DNA denaturation stage. One of the most significant advantages of LAMP is that it can be conducted at a stable temperature, for instance, in a dry block heater or an incubator. The products of LAMP can be detected much faster than in standard techniques, sometimes only requiring analysis with the naked eye. The following overview highlights the usefulness of LAMP and its effectiveness in various fields; it also considers the superiority of LAMP over PCR and presents RT-LAMP as a rapid diagnostic tool for SARS-CoV-2.

Expansion of Tick-Borne Rickettsioses in the World.

Tick-borne rickettsioses are caused by obligate intracellular bacteria belonging to the spotted fever group of the genus Rickettsia. These infections are among the oldest known diseases transmitted by vectors. In the last three decades there has been a rapid increase in the recognition of this disease complex. This unusual expansion of information was mainly caused by the development of molecular diagnostic techniques that have facilitated the identification of new and previously recognized rickettsiae. A lot of currently known bacteria of the genus Rickettsia have been considered nonpathogenic for years, and moreover, many new species have been identified with unknown pathogenicity. The genus Rickettsia is distributed all over the world. Many Rickettsia species are present on several continents. The geographical distribution of rickettsiae is related to their vectors. New cases of rickettsioses and new locations, where the presence of these bacteria is recognized, are still being identified. The variety and rapid evolution of the distribution and density of ticks and diseases which they transmit shows us the scale of the problem. This review article presents a comparison of the current understanding of the geographic distribution of pathogenic Rickettsia species to that of the beginning of the century.

Insights into the morphology and molecular characterisation of glacial relict Eurytemoralacustris (Poppe, 1887) (Crustacea, Copepoda, Calanoida, Temoridae).

Eurytemoralacustris (Poppe, 1887) is a stenothermic glacial relict whose narrow environmental requirements make it an indicator species for good ecological conditions. The primary threats to this species are eutrophication and global warming. Many authors have described E.lacustris in taxonomic keys; however, its morphological description is unsatisfactory. Therefore, in this study, we aimed to review morphological characteristics of E.lacustris that were previously undescribed in the literature and to provide the molecular characteristics based on the two conservative mitochondrial genes: cytochrome c oxidase I (COI) and cytochrome b (cytb). The new record of E.lacustris indicates that it is a more widespread species than previously hypothesized. Width-to-length ratio of the last female endopod segment of legs indicates variation among the widely distributed species of the genus in Europe (i.e., E.lacustris, E.velox (Lilljeborg, 1853), and E.affinis (Poppe, 1880)). We also found variability of number of setae on the second segment of male endopod. Furthermore, our analysis confirms the occurrence of species in different than exclusively freshwater habitats.

The effect of human sperm chromatin maturity on ICSI outcomes.

Because sperm chromatin may play a key role in reproductive success, we verify the associations between sperm chromatin abnormalities, embryo development and the ability to achieve pregnancy. The evaluation of sperm chromatin maturity using aniline blue (AB), chromomycin A3 (CMA3) and toluidine blue (TB) staining were carried out in group of males from infertile couples that underwent ICSI. Low levels of sperm chromatin abnormalities (< 16%) were found in most subjects (> 50%). A higher percentage of TB-positive sperm cells were discovered in the men from couples who achieved ≤ 50% fertilized oocytes compared to men who achieved > 50%. No significant differences were discovered by the applied tests between the men from couples who achieved ≤ 50% and those who achieved > 50% high-quality embryos on the 3rd or 5th day after fertilization, nor between the men from couples who achieved pregnancy and those who failed. The sperm chromatin maturity did not correlate with the ICSI results. However, the ROC analysis revealed a significant predictive value of TB-positive spermatozoa only for fertilization. Therefore, the TB assay can be considered as a useful test for the prediction of fertilization. Our findings suggest that the level of sperm chromatin abnormalities of the examined men was not clinically significant. No found associations between sperm chromatin maturity and embryo development and the ability to achieve pregnancy. We could not exclude the effects of the repairing processes in the fertilized oocyte. The use of complementary tests that verify the status of the sperm chromatin seems justified.

Molecular evidence for bacterial pathogens in Ixodes ricinus ticks infesting Shetland ponies.

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.

Genetic Variation in the ND1 Gene and D-loop in Protected and Commercially Exploited European Cisco (Coregonus albula L.) Populations.

The European cisco (Coregonus albula L.) is a species with high environmental requirements. The deterioration of environmental conditions in recent decades has decreased its distribution. Currently the species is conserved by stocking, and the few existing natural populations are at risk of extinction. Therefore, contemporary studies involve not only reporting phenotypic parameters, but also determining the genetic structure of the population. This is an important aspect monitored in the C. albula population, which provides information valuable for proper fishing economy. This study included valuable populations from lakes located in Drawa National Park (DNP) and Wigry National Park (WNP), as well as lakes used for commercial fishing. In order to molecularly characterize the European cisco, the control region and NDl gene were sequenced from 48 individuals from 9 populations from lakes throughout northern Poland. Analysis revealed that populations from two park lakes (Marta, Ostrowieckie) are unique. This was also the case for some sequences originating from Lake Wigry. The mean value of genetic diversity was 0.2% within each region and 0.1-0.3% between the investigated regions. The obtained results demonstrated the necessity to strengthen and protect natural populations of the European cisco, which constitute a valuable element of the European ichthyofauna.

Genotyping of Anaplasma phagocytophilum strains from Poland for selected genes.

Anaplasma phagocytophilum is a veterinary and medical tick-borne pathogen of vertebrates which invades granulocytes. The potential reservoirs of Anaplasma include game animals and small mammals. The aim ofthis study was to gain insight into the variability ofnucleotide sequences of ribosomal and selected protein-coding genes (ankA, msp4 and the groESL heat-shock protein operon) present in isolates of A. phagocytophilum collected from wild ruminants (Cervus elaphus and Capreolus capreolus) in Poland. Fragments of the genes were amplified using PCR and sequenced. High DNA variability was found in fragments of the msp4 and groESL genes (15 and 8 alleles, respectively), whereas ankA and 16S rRNA demonstrated very little or no variability (2 and 1 alleles, respectively). Unique allelic profiles were determined for the examined groups and allowed distinguishing 6 A. phagocytophilum genotypes, 2 of which were reported only in the roe deer (C. capreolus), and 1 in the red deer (C. elaphus).

Host and pathogen DNA identification in blood meals of nymphal Ixodes ricinus ticks from forest parks and rural forests of Poland.

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.

Use of DNA sequences for Rickettsia identification in Ixodes ricinus ticks: the first detection of Rickettsia monacensis in Poland.

The cosmopolitan tick Ixodes ricinus inhabiting Europe, including Poland, is a vector for many pathogens, such as various Rickettsia species, which spread to new territories. They are present mainly in the Mediterranean countries, but have also been found in Central Europe at increasing frequency. In the present study, the gltA gene, encoding citrate synthase, and an internal transcribed spacer (ITS) were employed to detect the DNA and identify the species of tick-borne pathogens of the Rickettsia genus. The presence of bacterial DNA was detected in 9.5% of the examined I. ricinus individuals. Based on the nucleotide sequences of the analysed genomic fragments, most pathogens were identified as Rickettsia helvetica, while Rickettsia monacensis was revealed in one case. We have described for the first time, to our knowledge, the occurrence of this species in Poland. Both markers employed in the experiments were successful in species identification of R. helvetica. The newly described species R. monacensis may be identified by the protein-coding gene, but the ITS nucleotide sequences proved insufficient.

Molecular evidence of vector-borne pathogens coinfecting dogs from Poland.

Ticks of the genus Ixodes are vectors for many pathogens, including Borrelia burgdorferi sensu lato, Anaplasma phagocytophilum and Rickettsia spp., and may also serve as vectors for Bartonella spp. However, the role of ticks in Bartonella transmission requires additional studies. The aim of this study was to investigate whether coinfection with two or more vector-borne pathogens can occur in the following three groups of dogs: I - dogs with suspected borreliosis (N = 92), II - dogs considered healthy (N = 100), and III - dogs with diagnosed babesiosis (N = 50). Polymerase chain reactions were performed to detect DNA of Anaplasma phagocytophilum, Rickettsia spp. and Bartonella spp. in the blood of dogs. In dogs of Group I, the DNA of both A. phagocytophilum and Bartonella sp. was detected (14% and 1%, respectively). In eight dogs, coinfection was indicated: A. phagocytophilum or Bartonella sp. with B. burgdorferi s.l. (the presence of antibodies against and/or DNA B. burgdorferi s.l.). In the case of five dogs positive for A. phagocytophilum DNA, no coinfection with B. burgdorferi s.l. was shown. In Group II, the DNA of A. phagocytophilum was detected in four dogs. In Group III, no pathogenic agents possibly transmitted by ticks were confirmed. No DNA of R. helvetica was detected in any of the groups studied.

[Detection of TBEV RNA in ticks as a tool for valuation of endemic area wide and sensitivity of TBE surveillance]. / Obecnosc RNA wirusa kleszczowego zapalenia mózgu w kleszczach Ixodes ricinus jako narzedzie oceny zasiegu obszarów endemicznych i czulosci nadzoru nad zachorowaniami na KZM.

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.

Detectability of tick-borne agents DNA in the blood of dogs, undergoing treatment for borreliosis.

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.

Symbiotic bacteria in oocyte and ovarian cell mitochondria of the tick Ixodes ricinus: biology and phylogenetic position.

Under natural conditions, eukaryote cells may contain bacteria. Arthropods such as ticks, insects or mites are a group particularly favoured by the obligate intracellular bacteria. While arthropods are vectors for some of them, other bacteria inhabit invertebrate host cells having entered mutualistic interactions. Such endosymbionts dwell usually in the host cell vacuoles or cytoplasm but have been also reported from tick oocyte mitochondria. The microorganisms contribute to mitochondria degradation, but their colonies are not eliminated from the tick cells affected. So far, such bacteria have been detected in three research centres. The Italian centre has reported on results of microscope and molecular analyses, while the Polish centre published molecular data. The Danish centre registered a 16S rRNA gene fragment in GenBank. Independent comparisons of the 16S rRNA gene sequences, carried out in the Italian and Polish centres, confirmed that the nucleotide sequences of the Ixodes ricinus endosymbionts formed a single clade with certain non-identified tick bacterium species isolated from the tick Haemaphysalis wellingtoni. On the other hand, pathogenic species of the genera Anaplasma, Ehrlichia and Rickettsia detected in I. ricinus as well as symbionts of the genus Wolbachia present in Culex pipiens and Drosophila simulans have been placed at a different site on the phylogenetic tree.

Molecular and serological diagnosis of Borrelia burgdorferi infection among patients with diagnosed Erythema migrans.

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 Bialystok, 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.

PCR detection of granulocytic Anaplasma and Babesia in Ixodes ricinus ticks and birds in west-central Poland.

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.

First detection of Anaplasma phagocytophilum in quill mites (Acari: Syringophilidae) parasitizing passerine birds.

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.

Borrelia burgdorferi sensu stricto in yellow-necked mice and feeding Ixodes ricinus ticks in a forest habitat of west central Poland.

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.