Prevalence of different Rickettsia spp. in Ixodes ricinus and Dermacentor reticulatus ticks (Acari: Ixodidae) in north-eastern Poland.

In two surveys conducted in April and May 2013, a total of 1148 unfed ticks belonging to two species, Ixodes ricinus and Dermacentor reticulatus, were collected by flagging lower vegetation in 15 different localities throughout the Bialowieza Primeval Forest (Podlaskie voivodship; north-eastern Poland) and in its buffer zone. In order to establish their infection rate with Rickettsia spp. individual adult ticks and pooled nymphs were tested by real-time PCR targeting the gltA gene. For the further identification of rickettsial species, positive samples were subjected for nested and semi-nested PCR targeting ompA and 16S rRNA genes, respectively, followed by sequencing analysis. Rickettsial DNA was detected in at least 279 ticks (minimum infection rate [MIR], 23.9%), including 52 nymphal and adult I. ricinus (MIR 8.6%) and 222 adult D. reticulatus (41%). Three species of SFG rickettsiae were identified: Rickettsia helvetica and 'Candidatus R. mendelii' in I. ricinus and R. raoultii in D. reticulatus and I. ricinus. Moreover, unidentified Rickettsia spp. which showed 99.4% identity, among others, with the uncultured Rickettsia sp. isolated from Cicadella viridis leafhopper, Rickettsia endosymbiont of Lasioglossum semilucens bee and R. bellii, were detected in I. ricinus, while Rickettsia sp. 98.3-98.4% homologous to Rickettsia secondary endosymbionts of Curculio spp. weevils was found in D. reticulatus. These results confirm the diversity of rickettsiae occurring in Poland. Further studies are needed to expand the knowledge on the species spectrum, prevalence and epidemiology of SFG rickettsiae in the country.

Kampinos National Park: a risk area for spotted fever group rickettsioses, central Poland?

Ixodid ticks are important vectors of a variety of bacterial and protozoan pathogens which cause infections in humans. In this study, altogether 1041 questing Ixodes ricinus (n = 305) and Dermacentor reticulatus ticks (n = 736), sympatrically occurring in Kampinos National Park (KPN), central-east Poland, were analyzed by PCR for Rickettsia species. Overall, the pathogen prevalence in ticks was 27.5 % for I. ricinus and 42.8 % for D. reticulatus. Sequencing analysis showed that the first tick species was exclusively infected with R. helvetica, whereas the latter was infected with R. raoultii. These organism may pose a threat for populations exposed to ticks. Preliminary results of a serosurvey of 74 KPN employees, inhabitants and visitors from the same area showed a 31.1 % total seroprevalence against SFG rickettsiae compared to 13.3 % seropositive blood donors of the control group. Risk factors significantly associated with IgG seropositivity were: occupational exposure to ticks (p = 0.002), frequency of tick bites (p = 0.02) and male gender (p = 0.005). Seropositive and seronegative individuals occupationally exposed to ticks did not differ significantly with respect to age and years of employment.

Different serotypes of dengue virus (DENV) imported by Polish travellers from dengue endemic areas to Poland.

BACKGROUND: Dengue viruses are the most widespread arboviruses (transmitted mainly by Aedes aegypti and Ae. albopictus mosquitoes), which have shown an unexpected geographic expansion. There are four dengue virus serotypes: DENV-1, DENV-2, DENV-3, and DENV-4. Subsequent infections increase the risk of developing severe dengue fever. MATERIALS AND METHODS: Samples from travellers returning from the endemic area of dengue fever were examined with the reverse transcription-polymerase chain reaction method. Primers amplified 743 bp fragment of the NS5 and 3'UTR genomic region of mosquito-borne flaviviruses of JEV group were used. RESULTS: The sequences from the 10 examined patients were compared to available DENV sequences in GenBank database and the basic local alignment search tool (BLAST) results confirmed that the infective virus was DENV-1 (6 patients), DENV-2 (2 patients) and DENV-3 (2 patients). CONCLUSIONS: For the first time in Poland, dengue virus serotypes were determined in travellers returning from dengue-endemic areas.

Detection and quantification of Anaplasma phagocytophilum and Babesia spp. in Ixodes ricinus ticks from urban and rural environment, northern Poland, by real-time polymerase chain reaction.

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

Wild boars as hosts of human-pathogenic Anaplasma phagocytophilum variants.

To investigate the potential of wild boars to host Anaplasma phagocytophilum, we analyzed bacterial 16S rRNA and ank genes. DNA sequencing identified several A. phagocytophilum variants, including a predominance of strains known to cause human disease. Boars are thus hosts for A. phagocytophilum, notably, strains associated with human granulocytic anaplasmosis.

[The occurrence of Cryptosporidium spp. in synanthropic flies in urban and rural environments]. / Wystepowanie Cryptosporidium spp. u muchówek synantropijnych na wybranych stanowiskach miejskich i wiejskich.

This study was carried out to determine the role of non-biting synanthropic flies as carriers of Cryptosporidium spp. oocysts in the vicinity of the city of Gdansk (NE Poland). In 2001-2003, flies were collected from three breeding sites: cow sheds and meadows in the Bystra cattle farm and municipal landfill Szadólki using inhaustors (aspirators) and entomologic nets. A total of 2358 specimens of the families: Muscidae (n = 1598), Calliphoridae (n = 739) and Sarcophagidae (n =21) were collected and analysed in 249 pools consisted of 9.5 insects, in average. Microscopic examination was used to detect Cryptosporidium spp. oocysts in the fly faeces deposited on the glass microscope slides and stained by Zhiel-Nielsen method. The mean number of faecal droplets per one glass slide was 11.5. Ooocysts of Cryptosporidium spp., stained from light pink to bright red, were found in fly faeces deposited on 25 (27.5%) of 91 glass slides checked. The highest prevalence of the pathogen was observed in faecal droplets deposited by flies collected in municipal landfill (50% investigated slides). DNA of Cryptosporidium spp. was extracted from the surface eluants of flies and/or their gut homogenates and purified. Then extracts were examined by PCR using CPB-DIAGF and CPB-DIAGR primers amplifying a variable region SSU-rRNA of all Cryptosporidium species. Altogether 387 isolates, 228 from surfaces and 159 from gut homogenates, were obtained from 249 pools of flies and analyzed. A specific 435 bp fragment of DNA was obtained in 49 (12.7%) lysates tested. In 10.4% pools, DNA of the pathogen was detected only in the surface eluants while in 7.6% only in gut extracts. In the case of two pooled samples (0.8%) Cryptosporidium spp. was found in both types of lysates. In total, Cryptosporidium spp. was detected in 47/249 pools of flies (18.9%). Assumed that each positive pool contained just one infected fly, the percentage of specimens able to oocysts transmission were calculated at the minimal level 2.0% (n = 47/2358). The result confirm that synanthropic flies can harbour oocysts of Cryptosporidium spp. both externally and internally, and disseminate them mechanically in the environment. Therefore, under unsanitary conditions they could be involved in the transmission of human and animal cryptosporidiosis.

Ixodes ricinus as a vector of Borrelia burgdorferi sensu lato, Anaplasma phagocytophilum and Babesia microti in urban and suburban forests.

In the suburban and urban forests in the cities of Gdansk, Sopot and Gdynia (northern Poland), Ixodes ricinus ticks should be considered as the vector of pathogenic microorganisms that may cause significant diseases in wild and domestic animals and humans. These microorganisms include etiologic agents of Lyme disease, human anaplasmosis (HA) and babesiosis: Borrelia burgdorferi sensu lato, Anaplasma phagocytophilum and Babesia microti, respectively. DNA extracts from 701 ticks collected in 15 localities were examined by PCR for the simultaneous detection of these 3 pathogens. Overall, 14 % were infected with A. phagocytophilum followed by 12.4 % with B. burgdorferi s.l. and 2.3 % with B. microti. In total, the percentage of infected females (32.9 %) was 2.4 times higher than in males (13.7 %) and 3.2 times higher than in nymphs (10.3 %). Among adult ticks (n = 303), 8.3 % were dually infected with A. phagocytophilum and B. burgdorferi s.l., 2.0 % with the agent of human anaplasmosis and B. microti and 0.3 % with borreliae and B. microti.

Human anaplasmosis in north-eastern Poland: seroprevalence in humans and prevalence in Ixodes ricinus ticks.

Sera of 500 inhabitants of north-eastern Poland, 450 suspected for Lyme borreliosis and 50 blood donors (control group), were analysed for the presence of IgG antibodies against Anaplasma phagocytophilum, the causative agent of human anaplasmosis (HA), known so far as human granulocytic ehrlichiosis (HGE). Forty one (9.1 %) sera of the study group and one serum (2 %) of the control group were positive using indirect fluorescence assay (IFA). The seropositivity tended to be more frequent among males (10.3 %) than females (7.6 %) and among the rural (10.3 %) than urban population (7.5 %); however, differences were of no statistical significance (p = 0.4). No age difference was found between the seropositive and the seronegative individuals (p = 0.77). The only factor increasing the risk of HA seropositivity found was forestry employment (p < 0.05). Additionally, a total of 559 Ixodes ricinus ticks, collected in the same area as sera, were investigated for the presence of A. phagocytophilum by the polymerase chain reaction (PCR) and 41 (8.7 %) of them were found to be positive. The infection level ranged from 2.3-13.7 %, depending on the area studied. Bacteria were significantly less frequently detected in nymphs - 2.1 % (5/235) than in adult ticks - 13.6 % (44/324) and in males--4.2 % (74/165) than in females--23.3 % (37/159) (p < or = 0.05). The obtained results confirm both the occurrence of HA foci in north-eastern Poland with I. ricinus as the principal vector of the A. phagocytophilum infection, and forestry workers as the main group at risk.

Cryptosporidium parvum and Giardia lamblia recovered from flies on a cattle farm and in a landfill.

Filth flies associated with a cattle barn and a municipal landfill were tested positive by combined immunofluorescent antibody and fluorescent in situ hybridization for Cryptosporidium parvum and Giardia lamblia on their exoskeletons and in their guts. More pathogens were carried by flies from the cattle barn than from the landfill; 81% of C. parvum and 84% of G. lamblia pathogens were presumptively viable.

Mechanical transmission of Cryptosporidium parvum oocysts by flies.

Long term field studies and laboratory experiments demonstrated that synanthropic filth flies can mechanically transmit infectious oocysts of Cryptosporidium parvum, an anthropozoonotic protozoan parasite which significantly contributes to the mortality of immunocompromised or immunosuppressed people. C. parvum oocysts are acquired from unhygienic sources, and can pass trough fly gastrointestinal track without alteration of their infectivity and can be subsequently deposited on visited surfaces. Transmission of the oocysts by adult flies occurs via: (1) mechanical dislodgement from the exoskeleton; (2) fecal deposition; and (3) regurgitation, i.e., vomits. Filth flies can cause human or animal cryptosporidiosis via deposition of infectious oocysts on the visited foodstuf, and the biology and ecology of synanthropic filth flies indicate that their potential for mechanical transmission of C. parvum is high.

[Acarofauna in the apartments of patients with perennial atopic rhinitis and functional tests of the respiratory system]. / Akarofauna mieszkan chorych na caloroczne atopowe zapalenie blony sluzowej nosa a badania czynnosciowe ukladu oddechowego.

Prolonged exposure to house dust mites (HDM) of the Pyroglyphidae family leads to the development of perennial allergic rhinitis (PAR) in sensitized subjects. In PAR patients bronchial hyperreactivity--the clinical manifestation of allergic inflammation in the lower airways is often observed. The aim of the study was to estimate the influence of the exposure to HDM in PAR patients on selected functional parameters of the lower airways. The study was performed on 64 patients with PAR caused by allergy to HDM (Dermatophagoides pteronyssinus, Dermatophagoides farinae) without either symptoms of asthma or spirometry abnormalities. The lower airways function was estimated by bronchial histamine provocation test (BHR) and flow-volume spirometry. The house dust mite quantity was measured in dust samples collected in spring and autumn. One sample was assessed by flotation technique with the use of methylene chloride and the mites species were identified (direct method) and the other sample was used to perform the Acarex test (semiquantitative guanine determination test). Positive result of Acarex test was obtained in spring in 72% houses and in autumn in 63%. The predominance of the Pyroglyphidae family was observed in PAR patients' houses. The study showed that the exposure to the allergenic HDM in the quantity higher than 0.6 mg guanine/g dust in autumn implies the increased incidence of both bronchial hyperreactivity and decreased airflow in the small bronchi in control spirometry tests. A strict correlation between the quantity of mites in houses in spring and autumn measured by the direct (flotation method) and the indirect (Acarex) tests was observed. Both methods proved to be equivalent in detecting allergens of house dust mites.

Coinfection of Ixodes ricinus (Acari: Ixodidae) in northern Poland with the agents of Lyme borreliosis (LB) and human granulocytic ehrlichiosis (HGE).

Adult Ixodes ricinus ticks were collected from Pomerania province, northern Poland, to determine the presence of infection with agents of human granulocytic ehrlichiosis (HGE) and Lyme borreliosis by using the polymerase chain reaction (PCR). Of the 424 ticks 19.2% and 11.6% contained ehrlichiae and spirochetes, respectively. Frequency of single infection with the HGE agent was 63/424 while frequency of single infection with Borrelia burgdorferi sensu lato was 28/424. As many as 21/424 ticks (5%) contained both pathogens. This finding supports suggestions that both HGE agent and B. burgdorferi s. l. perpetuate in the same foci and frequently co-infect the same tick vector thereby increasing the risk of humans acquiring mixed infection.

Prevalence of Borrelia burgdorferi sensu lato in the selected Ixodes ricinus (Acari: Ixodidae) population in Weilburg forests, Hesse, Germany.

We studied the presence of Borrelia burgdorferi sensu lato in Ixodes ricinus ticks collected in woodland areas in the vicinity of Weilburg (Hesse, Germany). Ticks (n = 13,347) were collected in five localities in 1996 and were fixed in 70% ethanol. Out of them, 4404 specimens were analysed individually (n = 406) and in 796 pools of 2-10 specimens (n = 3998) by the polymerase chain reaction (PCR). The calculated minimal infection rate of individual and pooled ticks was 0% in larvae (n = 280), 6.9% in nymphs (n = 3600), 13.9% in males (n = 280) and 20.9% in females (n = 244). Prevalence of B. burgdorferi s. l. in I. ricinus was determined with respect to the abundance and seasonal activity of the ticks.