Morphometric analysis - effect of the radiofrequency interface of electromagnetic field on the size of hatched Dermacentor reticulatus larvae.

INTRODUCTION AND OBJECTIVE: Electromagnetic radiation interactions with living systems have been one of determining factors in biological evolution. This study investigates the effect of 900 MHz radiofrequency (RF) electromagnetics field (EMF) exposure of eggs on the development of Dermacentor reticulatus larvae. The basic objective was to determine whether the 900 MHz RF-EMF has the potential to influence the size of the body of the hatched larvae of D. reticulatus ticks. MATERIAL AND METHODS: To this aim, eggs from 3 fully engorged females of D. reticulatus were included in the test procedure. Altogether four groups of eggs were designated which included eggs from each female. We used RF-EMF frequency of 900 MHz. Eggs were exposed to EMF for different time periods (30, 60 and 90 minutes) in dark, electromagnetically shielded anechoic chamber. After the irradiation eggs were allowed to hatch in climatic chamber. Randomly selected 200 larval individuals were measured to get basic morphological records. Four body traits including the total body length (TBL), length of gnathosoma with scutum (GSL), the total body width (TBW), and the width of basis capituli (BCW) were measured. RESULTS: The D. reticulatus larvae hatched from eggs exposed for 60 minutes, had demonstrably larger dimensions of all measured body traits not only as a control unexposed group but also as other experimental groups. CONCLUSIONS: The study shows, particularly, that artificial EMF that is used in smartphone technology impacts seriously D. reticulatus larvae development.

Does Traditional Feeding of Outdoor Guard Dogs Provide a Food Resource for Wild Mammals and Birds?

Access to food is crucial in the life of birds and affects reproduction, survival and, consequently, population size. In the case of bird species inhabiting villages, poorer food conditions now exist, mainly because of changes in the lifestyle of rural residents and a reduction in the number of farm animals traditionally housed in backyards. Recent changes have also affected dog populations in villages, and the majority of them are no longer kept outside as guard dogs, but rather inside houses as pets. We investigated how traditional care of dogs impacted rural birds and other animal populations. The study was carried out at the end of winter and early spring in 29 farmsteads in western Poland. Using camera traps, it was found that the food fed to dogs was also taken by seven species of birds and at least three species of mammals. The most numerous species taking dog food was the house sparrow, Passer domesticus, which is declining in Europe. In the case of this species, females were more likely than males to use food given to dogs, with a clear preference for food prepared in the human kitchen. We conclude that the food provided to domestic pets can be an important component of the diet of wild birds and mammals living close to humans.

Similar Trajectories in Current Alcohol Consumption and Tick-Borne Diseases: Only Parallel Changes in Time or Links Between?

In a modern world, both tick-borne diseases and alcohol consumption are among major public health threats. In the present opinion article, we pose the question, whether these two health problems: alcohol consumption and tick-borne diseases prevalence can be related. We hypothesize that it is possible due to at least three factors: outdoor places chosen for alcohol consumption, behavioral changes induced by alcohol, and possible stronger attraction of human hosts after alcohol consumption to ticks. Many important clues are coming from social studies about people's preference of places to consume alcohol and from studies regarding the attraction of people consuming alcohol to mosquitos. These data, however, cannot be directly transferred to the case of alcohol consumption and ticks. Therefore, we suggest that more detailed studies are needed to better understand the possible individual attractiveness of people to ticks and ways alcohol may influence it.

Emergence of tick-borne pathogens (Borrelia burgdorferi sensu lato, Anaplasma phagocytophilum, Ricketsia raoultii and Babesia microti) in the Kyiv urban parks, Ukraine.

To date, only limited data about the presence of ticks and circulation of tick-borne pathogens in urban parks of Kyiv in northern Ukraine are available. In total, 767 ticks (696 Ixodes ricinus and 69 Dermacentor reticulatus) collected in seven urban parks and one suburban oak wood park in Kyiv were individually analyzed by the PCR assays. Tick-borne pathogens, namely spirochetes from Borrelia burgdorferi sensu lato complex, Anaplasma phagocytophilum, and Babesia microti, were detected in 11.1% of tested I. ricinus ticks. In total, 4% of I. ricinus ticks tested positive for the presence of B. burdorferi s.l. (Borrelia afzelii and Borrelia garinii), 5.2% for A. phagocytophilum, and Ba. microti was confirmed in 1.9% of examined ticks. Mixed infections were recorded in four DNA samples, representing the prevalence of 0.6%. One female and two I. ricinus nymphs were simultaneously infected with B. afzelii and A. phagocytophilum, and one female carried B. afzelii and Ba. microti. In addition, 10.1% of D. reticulatus ticks tested positive for Rickettsia raoultii. Identification of infectious agents and their diversity, assessment of the relative epidemiological importance and determination of the prevalence in questing ticks from central parts of the cities are crucial steps towards the tick-borne diseases surveillance in urban environment.

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

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

First molecular identification of Babesia gibsoni in dogs from Slovakia, central Europe.

Canine babesiosis is a severe and potentially life threatening infection. In Europe, Babesia canis is considered to be the most common species responsible for the disease. We report two cases of babesiosis caused by Babesia gibsoni. The polymerase chain reaction, restriction fragment length polymorphism analysis and further sequencing of 18S rRNA gene fragments from blood samples of both dogs revealed the identity of isolates with B. gibsoni genotypes from other dogs worldwide. This species was previously not known to infect dogs in Slovakia. It is resistant to traditional anti-babesial therapy. Therefore, correct diagnosis is crucial for the successful treatment, especially in dogs with hemolytic anemia and febrile conditions.

The infection of reintroduced ruminants - Bison bonasus and Alces alces - with Anaplasma phagocytophilum in northern Poland.

The north-eastern part of Poland is considered an area of high risk for infection with tick-borne diseases, including with human granulocytic ehrlichiosis (HGE) agents. The etiological agent of HGE is Anaplasma phagocytophilum. As the animal reservoir for A. phagocytophilum in the environment serve the species from Cervidae and Bovidae families. European bison (Bison bonasus) and elk (Alces alces) are the big ruminant species, reintroduced to the forests of Middle Europe after many decades of absence. In the foci of zoonotic diseases they are able to play a role as natural reservoir to pathogens, however, their status as protected animals means their study has been rare and fragmentary. The studies of B. bonasus were conducted in Bialowieza Primeval Forest and A. alces in Biebrza National Park. PCR amplifications were performed using primers amplifing the end of the groES gene, the intergenic spacer and approximately two-thirds of the groEL gene in the first round, and primers that span a 395-bp region of the groEL gene were used in the second round. The positive results were obtained in B. bonasus and A. alces, the prevalence of infection was 66.7 and 20.0%, respectively. Randomly selected samples were sequenced, sequences were compared with GenBank entries using Blast N2.2.13 and determined as A. phagocytophilum. The results presented herein are the first record of the presence of Anaplasma phagocytophilum in A. alces, and at the same time confirm the previous observations regarding the infection of B. bonasus with A. phagocytophilum.

New records of spotted fever group rickettsiae in Baltic region.

Tick-borne rickettsiae are considered to be emerging, but there is still a lack of data on the occurrence and prevalence of the spotted fever group rickettsiae across Europe, especially in the Baltic countries. The aim of the present study was to investigate the presence of Rickettsia spp. in Dermacentor reticulatus and Ixodes ricinus ticks, as well as to determine their prevalence in various regions of Lithuania and Latvia. The prevalence of Rickettsia spp. in geographically distinct populations of D. reticulatus on transect Baltic-Adriatic regions was compared. The molecular analyses of 1859 D. reticulatus and 361 I. ricinus from 48 localities in Lithuania and Latvia revealed the presence of Rickettsia raoultii in D. reticulatus and Rickettsia helvetica in I. ricinus. The prevalence of pathogens in D. reticulatus (4.9%) and I. ricinus (17%) ranged in different locations from 0% to 36.9% and 0%-31.3%, respectively. Higher overall infection rates were detected in D. reticulatus collected in Poland (15.8%) and Slovakia (11.4%), with the prevalence range in different locations from 0% to 59.6%. Our study is the first demonstration of the presence of R. raoultii in D. reticulatus ticks in the Baltic countries and R. helvetica in questing I. ricinus ticks in Lithuania.

The infection of questing Dermacentor reticulatus ticks with Babesia canis and Anaplasma phagocytophilum in the Chernobyl exclusion zone.

Tick occurrence was studied in the Chernobyl exclusion zone (CEZ) during the August-October 2009-2012. Dermacentor reticulatus ticks were collected using the flagging method and then screened for infection with Anaplasma phagocytophilum and Babesia canis by a PCR method incorporating specific primers and sequence analysis. The prevalence of infection with B. canis canis and A. phagocytophilum was found to be 3.41% and 25.36%, respectively. The results present the first evidence of B. canis canis and A. phagocytophilum in questing D. reticulatus ticks from the Chernobyl exclusion zone. They also reveal the presence of tick-borne disease foci in areas with no human activity, and confirm that they can be maintained in areas after a nuclear disaster with radioactive contamination.

Anaplasma infections in ticks and reservoir host from Slovakia.

Anaplasma phagocytophilum is a worldwide distributed bacterium with a significant medical and veterinary importance. It grows within the phagosome of infected neutrophils and is responsible for human granulocytic anaplasmosis (HGA), tick-borne fever (TBF) of small ruminants and cattle, canine and equine granulocytic anaplasmosis, but infects also a great variety of wildlife species. Wild ungulates and rodents are considered reservoirs of infection in natural foci. The objective of this study was to determine the spectrum of animal species involved in the circulation of A. phagocytophilum in Slovakia and to analyze the variability of obtained nucleotide sequences, in order to determine whether genotypes from Slovakia cluster according to host-species or geographical location. Several animal species and vector ticks were screened for the presence of members of the family Anaplasmataceae using PCR based methods. Additional data on the molecular evidence of Anaplasma ovis and Candidatus Neoehrlichia mikurensis are presented. These pathogens were detected in tested sheep flocks and rodents with the mean infection rates of 8.16% and 10.75%, respectively. A. phagocytophilum was genotyped by 16S rRNA and groEL gene sequencing. Bacterial DNA was confirmed in questing ixodid ticks, in domesticated canine, wild rodents and several species of wild ungulates. In European isolates, 16S rRNA gene does not seem to be an appropriate locus for the analyses of heterogeneity as it is too conservative. Similarly, 16S rRNA isolates from our study did not reveal any polymorphisms. All isolates were identical in overlapped region and showed identity with sequences from ticks, horses or ruminants previously isolated elsewhere in the world. On the other hand, the groESL heat shock operon is widely used for determination of diversity and the analyses have already revealed considerable degree of heterogeneity. Tested ungulates were infected with A. phagocytophilum to a considerable extent. High proportions of red and roe deer tested positive and the rates of infection reached over 60.0%. GroEL sequences from canine, wild ungulates and ticks from Slovakia clustered within a clade together with isolates from horses, humans, wild ungulates and ticks from Slovakia or elsewhere in the world. Sequences from rodents clustered apart from those obtained from wild ungulates, ticks and humans. These results suggest that European rodents do not harbour A. phagocytophilum strains with strong zoonotic potential such as those from United States.

Ixodes ricinus abundance and its infection with the tick-borne pathogens in urban and suburban areas of Eastern Slovakia.

BACKGROUND: Raising abundance of ticks and tick-borne diseases in Europe is the result of multiple factors including climate changes and human activities. Herein, we investigated the presence and seasonal activity of Ixodes ricinus ticks from 10 urban and suburban sites in two different geographical areas of southeastern and northeastern Slovakia during 2008-2010. Our aim was to study the abundance of ticks in correlation with the environmental factors and their infection with Borrelia burgdorferi sensu lato, Anaplasma phagocytophilum and Neoehrlichia mikurensis. METHODS: Questing I. ricinus ticks were collected from ten urban and suburban sites in Eastern Slovakia. A total of 670 ticks were further analysed for the presence of B. burgdorferi s.l., A. phagocytophilum and N. mikurensis by molecular methods. Tick site and environmental relations were analysed using General Linear Models (LM). The differences between the number of Lyme borreliosis cases between the Kosice and Bardejov regions during a ten-year period were tested by Wilcoxon matched pairs test. RESULTS: In total, 2921 (1913 nymphs, 1008 adults) I. ricinus ticks were collected from 10 study sites during the main questing season. Tick activity and relative abundance differed between locations and months. Temperature and humidity were the main factors affecting the tick abundance and questing activity. Out of 670 examined ticks, 10.15% were infected with spirochetes from B. burgdorferi s.l. complex (represented by B. afzelii, B. garinii, B.valaisiana and B. burgdorferi s.s.), 2.69% with the A. phagocytophilum and 2.39% with N. mikurensis. The number of Lyme borreliosis cases per 100,000 inhabitants in the Bardejov region was significantly higher than in the Kosice region. CONCLUSIONS: Our data indicate that the risk of infection with tick-borne pathogens in Eastern Slovakia is common since 15.2% of ticks were infected at least with one of the tested microorganisms. Even though the abundance of ticks was affected by the microclimatic conditions and the prevalence of pathogens differed between the habitats, the infection risk for humans is also affected by human activities leading to an increased contact with infected ticks.

Emergence and genetic variability of Anaplasma species in small ruminants and ticks from Central Europe.

Anaplasmoses are common tick-borne zoonotic bacterial diseases of livestock and free-living ungulates from the genus Anaplasma that are recently emerging in Central Europe. The main aim of this study was to analyze the prevalence and genetic variability of Anaplasma phagocytophilum and Anaplasma ovis in small ruminants and questing ticks from six different sites in Slovakia and the Czech Republic using the PCR of the msp4 gene followed by the sequence analysis. At two farms from southeastern Slovakia, 66.1% small ruminants were infected with A. ovis in contrast to one positive animal from both sites in northern Slovakia. It was represented by two different genotypes. A. phagocytophilum was present in all tested flocks with the infection prevalence ranging from 0.9% to 5.7%. None of the tested questing ticks carried A. ovis. A. phagocytophilum was detected in 1.1% and 7.8% of questing Ixodes ricinus ticks collected around the farms located in southeastern and northern Slovakia, respectively. A. phagocytophilum revealed higher intraspecific diversity than A. ovis.

First case of human granulocytic anaplasmosis from Slovakia.

In order to evaluate the potential risks of human granulocytic anaplasmosis in Slovakia, blood and serum samples of hunters and foresters from the northern parth of Slovakia were tested. We present the first case of HGA from Slovakia confirmed by nested PCR amplification of the 16S rRNA gene fragment of A. phagocytophilum.

Climate warming and tick-borne encephalitis, Slovakia.

Increased tick-borne encephalitis (TBE) cases have been reported in central Europe. To investigate temporal trends in the altitude at which TBE cases occur in Slovakia, we analyzed the number of TBE cases during 1961-2004. Since 1980, TBE cases moved from lowlands to submountainous areas, most likely because of rising temperature.

Spatial distribution of Dermacentor reticulatus tick in Slovakia in the beginning of the 21st century.

A new field survey monitoring the spatial distribution of Dermacentor (D.) reticulatus (Fabricius, 1794) tick in Slovakia was carried out in 2005-2008 in order to record changes in its distribution when compared to former studies. Last surveys on the geographical distribution were conducted in 1950s and 1970s and the presence of D. reticulatus was determined along the rivers in the south-east (Latorica) as well as in the south-west (Morava, Dunaj) Slovakia. In the present survey new areas with D. reticulatus occurrence were detected, providing evidence that this tick species has extended its range in the surroundings of its former habitats but also by at least 200 km further North and by 300 m of elevation into higher altitudes. D. reticulatus is known to transmit Babesia spp. causing babesiosis in cattle and dogs. Expansion of D. reticulatus range is therefore likely to bring a spread of babesiosis, which can be severe or fatal especially for dogs.

Anaplasma phagocytophilum infection of red foxes (Vulpes vulpes).

The investigation of Anaplasma phagocytophilum infection covered 111 red foxes from the Mazovian Province. Determination was based on PCR amplification of 16S rRNA gene and the portion of msp4 gene coding MSP4 major surface protein. The presence of A. phagocytophilum was found in 2.7 % of foxes.

Evaluation of recombinant line immunoblot for detection of Lyme disease in Slovakia: comparison with two other immunoassays.

In the present study the sensitivity and the specificity of three serological tests (enzyme-linked immunosorbent assay [ELISA], indirect fluorescent antibody test [IFA], and recombinant line immunoblot) were compared by examining 74 sera from patients diagnosed with Lyme disease in Eastern Slovakia. In addition, the reactivity to each of the recombinant proteins in the immunoblot was examined in order to evaluate their diagnostic value. Generally, the immunoblot (93.2%) and the ELISA (90.5%) were significantly more sensitive than the IFA (64.9%; df = 1; p < or = 0.001). Correlation between results of the ELISA, IFA, and immunoblot for IgM or IgG, when two tests were always compared, one to the other, ranged from r(s) = 0.673 to r(s) = 0.905. In the immunoblot, the highest sensitivity was observed in DbpA and VlsE proteins (76.9% and 84.6%, respectively) in IgG testing of the sera from the patient group of Lyme arthritis. VlsE proteins, together with OspC proteins, were also shown to be useful for IgM antibody detection in erythema migrans patients (up to 44.4% and 53.7% sensitivity, respectively). Our results indicate that both the ELISA and the recombinant immunoblot test were more satisfactory for seroconfirmation of Lyme disease than IFA. Moreover, the reseach confirmed diagnostic value of the in-vivo expressed proteins (VlsE and DbpA), which might have the potential to play an important role in improving whole-cell antigen-based testing.

Immune complexes in early Lyme disease.

The study investigated the presence of Borrelia-specific antibodies captured in immune complexes (ICs) in patients with early Lyme disease manifested by erythema migrans. Out of 18 patients, 15 (83.3%) tested positive for polyethylene glycol-precipitated ICs containing IgM antibodies, while only 4 (22.2%) were IgG positive. These results are in accordance with our findings obtained by standard ELISA and recombinant blot, which indicated that ICs might be used for serological diagnosis of the early disease.

Hepatozoon canis infection in Slovakia: imported or autochthonous?

Tissue samples from nine red foxes (four samples of striated muscle tissue and five samples of heart tissue) that originated from the Michalovce district (Slovakia), an area with endemic occurrence of canine babesiosis were examined by PCR method using primers amplifying a fragment of the 18S rRNA spanning the V4 region of Babesia and Theileria. An unexpected determination of 450 bp DNA fragment of Hepatozoon canis was found in four samples. Partial sequences of the 18S rRNA gene from the H. canis showed 100% similarity with the sequence from Brasil isolate of H. canis from a pampas fox (Pseudalopex gymnocercus) (AY471615) as well as from a fox in Spain (AY150067) and from a dog in Brazil (AY864677). In the present study, we report the first PCR detection of Hepatozoon canis in a naturally infected red fox from Slovakia, a Rhipicephalus sanguineus-free region. We assume that the infection was spread by infected R. sanguineus that might have been brought to Slovakia by travelers, by golden jackals, or by foxes migrating because of expansion of golden jackals and environmental and climate changes.

Prevalence of Borrelia burgdorferi s.l. OspA types in Ixodes ricinus ticks from selected localities in Slovakia and Poland.

In this study, 746 questing Ixodes (I.) ricinus ticks from eastern Slovakia and 187 ticks from southern Poland were investigated for infection with Borrelia (B.) burgdorferi sensu lato and different outer surface protein A (OspA) types by an improved restriction fragment length polymorphism (RFLP) analysis of the ospA gene. The method enables differentiation of both single and multiple infections with B. burgdorferi s.s. (OspA type 1), B. afzelii (OspA type 2), B. garinii (OspA types 3-8), B. valaisiana (subgroups I and II), B. lusitaniae, B. bissettii, and the recently described genospecies A14S. Broad heterogeneity in B. burgdorferi s.l. was found including all species and subtypes except for B. lusitaniae, B. bissettii, and genospecies A14S. Regional prevalence of B. burgdorferi s.l. varied between 8% and 22.5%. The most frequent species were B. garinii (45.4%) and, notably, B. burgdorferi s.s. (31.3%). I. ricinus nymphs harbored almost exclusively B. burgdorferi s.s. and B. garinii OspA type 4, while in adults a broad variety of B. burgdorferi s.l. types was found. Mixed infections were significantly more often in nymphs than in adult ticks. In all mixed infected nymphs, B. burgdorferi s.s. with OspA type 4 was present. These data strongly suggest that B. burgdorferi s.s. and B. garinii OspA type 4 are maintained in these areas by specific transmission cycles involving a so far undetermined vertebrate host which is frequently fed on by I. ricinus larvae. This improved method provides a reliable tool for epidemiological studies on the heterogeneity of B. burgdorferi species and OspA types, an important prerequisite for improved local risk assessment and for test- and vaccine development for Europe.