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In central Europe, at least three flaviviruses circulate among vectors and vertebrate hosts. West Nile virus (WNV) and Usutu virus (USUV) are mosquito-borne viruses maintained in the nature by enzootic cycle between mosquitoes and birds. Tick-borne encephalitis virus (TBEV) is a flavivirus causing annual human cases in Slovakia. The aim of this study is the prevalence assessment of flavivirus infections in horses (n = 145) and birds (n = 109) by enzyme-linked immunosorbent assay (ELISA) and confirmation by neutralization test (VNT). WNV antibodies have been detected in 11.7% of tested horses and 11.9% of tested birds and confirmed in 6.9% of horse and 9.2% of bird samples. None of the WNV seropositive or dubious horses had WNV IgM (ELISA), and none of the tested horses had USUV neutralizing antibodies. Autochthonous WNV infections have been confirmed in 16.7% of horses without international travelling history. Most of them were from western Slovakia with known endemic WNV transmission. An autochthonous WNV infection in a horse from highland area of Kremnické vrchy (central Slovakia) with unknown data of WNV circulation and without travelling history was detected. TBEV antibody was detected in 6.2% of horses and in 3.4% has been confirmed. In two horses, WNV and TBEV infection could not be distinguished. Confirmed WNV seropositive were eight raptors showing nonspecific signs or suffering from trauma, one white stork, and one house sparrow. The sparrow was caught in a locality in eastern Slovakia, where WNV RNA had been previously detected in sparrows. USUV neutralizing antibodies were present in pooled sample from four Eurasian great tits. Because of insufficient volume, TBEV VNT was not carried out in birds. Results further prove the endemicity of WNV and other vector-borne flaviviruses in natural and accidental hosts in Slovakia, giving better insight in flavivirus epidemiology in European countries in general.

Based on initial reports of infection with Francisella tularensis in dogs in northern Europe, a serological study in hunting dogs was conducted in Austria. Five (6.25%) tested positive. The results highlight the potential for disease transmission to dogs and raise the question of whether dogs could be a source of infection for humans.

Introduction: Kurdistan Province in Iran is a historical focus for plague and tularemia. This study aimed at assessing the current status of these two foci by studying their rodent reservoirs., Materials and Methods: Rodents were trapped and their ectoparasites were collected. The genus and species of both rodents and ectoparasites were determined. Serological analyses of rodent blood samples were done by enzyme-linked immunosorbent assay for plague and by standard tube agglutination assay for tularemia. Rodent spleen samples were subjected to bacterial culture, microscopic examination, and real-time PCR to search for active plague or tularemia infection., Results: During this study, 245 rodents were trapped, of which the most abundant genera were Apodemus (40%), Mus (24.49%), and Meriones (12.65%). One hundred fifty-three fleas, 37 mites, and 54 ticks were collected on these rodents. The results of all direct and indirect tests were negative for plague. Serological tests were positive for tularemia in 4.8% of trapped rodents., Discussion: This study is the first report on the presence of tularemia infection in rodents in Western Iran. Since Meriones persicus is a known reservoir for plague and tularemia, and this rodent carried plague and tularemia vectors in Marivan and Sanandaj districts, there is a real potential for the occurrence of these two diseases in this region.

Crimean-Congo hemorrhagic fever virus (CCHFV) is a typical tick-borne pathogen that causes an increasing number of severe infections in many parts of Africa, Asia, the Middle East, and the Balkans, as well as in some other parts of Europe. The virus is transmitted primarily by Hyalomma spp., and the spectrum of natural hosts for CCHFV is broad, including wild and domestic animals. Although, the presence of CCHFV was hypothesized in Hungary, no significant research activity has been carried out in the past 30 years. In the present study, we provide serological evidence of CCHFV infection in Lepus europeus using newly developed antibody detection assays. Of 198 samples, 12 (6%) were positive for immunoglobulin G antibody against CCHFV, with 2 independent detection assays. This observation indicates a need for a large-scale surveillance to estimate the potential public health risk of CCHFV in Hungary.

Several new taxa belonging to the genus Francisella have been described recently. The present study describes the prevalence of Francisella tularensis and Francisella-like endosymbionts (FLE) in ticks collected from Hungary from 2007 to 2009 and characterizes the genetic variability of FLEs. A total of 5402 Ixodid ticks (Ixodes ricinus, I. acuminatus, Dermacentor marginatus, D. reticulatus, Haemaphysalis inermis, H. concinna, H. punctata) were collected from vegetation and animal hosts and tested with conventional PCR, detecting the 16S rRNA and tul4 genes. F. tularensis ssp. holarctica was found in 2 pools of H. concinna and 1 pool of D. reticulatus, both representing minimum prevalence (calculated with 1 infected tick per pool) of 0.27% whereas the sequences of a FLE were detected in 11 pools of D. reticulatus showing a minimum prevalence of 3%. Although the tul4 gene sequence of this FLE was identical to all Hungarian and Portuguese FLEs found earlier, and the 16S rRNA sequence was also identical to the sequence of the endosymbiont of D. reticulatus described in Bulgaria, these 16S rRNA gene coding sequences differed in 2 nucleotides from the one found earlier in this tick species in Hungary. This divergence may appear to be a minor difference between the sequences, potentially even resulting from a technical failure, but it could also indicate a significant difference stemming from the conservative genetic character of Francisellaceae. Thus, it raises a question about the number of FLE variants circulating in D. reticulatus in Europe and indicates the need for further data about the FLEs described in other parts of the continent and new FLE genotyping markers.

The aim of this study was to analyze synanthropic birds as risk factors for introducing ticks and tick-borne pathogens into human settlements, with an emphasis on rickettsiae. Altogether 184 subadult ticks were found on 5846 birds. Tick infestation was most prevalent during the spring. In this sample group the majority of ticks were molecularly identified as Ixodes ricinus, and three individuals collected from the European robin as Hyalomma marginatum marginatum. The latter is the first molecularly confirmed occurrence of this species in Hungary. Rickettsia aeschlimannii was detected in H. marginatum, also for the first time in Hungary, and in ticks from an urbanized bird species north of the Mediterranean countries. The overall prevalence range of rickettsiae (including R. helvetica and R. monacensis) in ticks of synanthropic birds was 29-40%, exceeding that in questing ticks of relevant species reported earlier. Additionally, in specimens of I. ricinus, the presence of Borrelia burgdorferi sensu lato (s.l.), Anaplasma phagocytophilum, and a new Francisella-like genotype was also verified. Thus, it can be concluded that birds with urban or periurban habitats pose a high risk as tick carriers and reservoirs of zoonotic agents, especially of rickettsiae.

Q fever is an important zoonotic disease caused by Coxiella burnetii. There are few reliable data about C. burnetii infection available. The aim of this study was to assess the importance and potential infectious sources of Q fever in Hungary. A total of 215 milk samples (10 individual samples from each herd and 1 bulk tank milk sample from each cattle herd), and 400 serum samples (20 from each herd) were tested from 15 dairy cattle herds and 5 sheep flocks located in different parts of Hungary. The study found 19.3% (58/300) and 38.0% (57/150) seropositivity in cattle, and 0% (0/100) and 6.0% (3/50) seropositivity in sheep, by complement fixation test (CFT) and enzyme-linked immunosorbent assay (ELISA), respectively. C. burnetii DNA was detected by IS1111 element-based TaqMan real-time polymerase chain reaction (PCR) in 8.7% (13/150) of individual dairy cow milk samples, 4.0% (2/50) of individual sheep milk samples, and 66.7% (10/15) of dairy bulk tank milk samples. Samples taken from nine different commercially-available pasteurized cow milk products from different Hungarian producers were also tested for the presence of C. burnetii DNA, and eight of these samples were found to be positive (88.9%). The real-time PCR examination of 5402 ixodid ticks collected from different parts of the country yielded negative results. Knowledge of the true prevalence of Q fever is crucial for policymakers involved in evidence-based decision making.

The aim of our study was to investigate the presence of Borrelia burgdorferi sensu lato (s.l.) and Anaplasma phagocytophilum in small mammals and ticks using polymerase chain reaction and to gain information about the prevalence and possible coexistence of these pathogens at a selected site in Hungary. Two hundred seventy-seven small mammals were trapped in South-Eastern Hungary during 2009. Tissue samples and a total of 831 ectoparasites (Ixodes ricinus, Ixodes acuminatus, Haemaphysalis concinna, Ctenophtalmus assimilis, and Nosopsyllus fasciatus) were collected from small mammals. One thousand one hundred and six I. ricinus and 476 H. concinna were collected from the vegetation during the investigation. Neither A. phagocytophilum nor B. burgdorferi s.l. was detected in any of the mammal tissue samples. A. phagocytophilum was not found in ticks collected from small mammals. Very low minimum prevalence was found for all pathogens (0.62% for Borrelia afzelii in ticks collected from small mammals, and 0.57%, 0.06%, and 0.19% for A. phagoctyophilum, B. afzelii, and Borrelia garinii, respectively, in questing ticks). The present study is the first report of borreliae from I. acuminatus and H. concinna from Hungary.

A 1-year study of the ecological cycle of Francisella tularensis was performed in an enzootic area during an inter-epizootic period. The study was based on multiple sampling of all major constituents of the disease cycle. Seroprevalence of tularemia in the European brown hare (Lepus europaeus) population was 5.1% (10/197) with low antibody titers (1/10 and 1/20), and F. tularensis ssp. holarctica was isolated from four hares. F. tularensis was not detected in the 38 common voles (Microtus arvalis), 110 yellow-necked mice (Apodemus flavicollis), or 15 stripped field mice (Apodemus agrarius) trapped during the study, or the by-catch of 8 Eurasian pygmy shrews (Sorex minutus) or 6 common shrews (Sorex araneus). A total of 1106 Ixodes ricinus and 476 Haemaphysalis concinna ticks were collected from vegetation, and 404 I. ricinus, 28 H. concinna ticks, and 15 Ctenophtalmus assimilis and 10 Nosopsyllus fasciatus fleas were combed off small mammals. One H. concinna female and one nymph collected from the vegetation was found infected with F. tularensis ssp. holarctica by TaqMan polymerase chain reaction, thus resulting a 0.42% (2/476) prevalence. F. tularensis-specific DNA was not detected in environmental water samples, and the examined 100 sheep, 50 cows, and 50 buffalos grazed at the study area were all seronegative. During inter-epizootic periods, F. tularensis ssp. holarctica seems to persist only in the European brown hare--H. concinna cycle at the studied habitat. H. concinna may not serve exclusively as an arthropod vector, but it may also harbor bacteria for 3-4 years through multiple life stages and act as an important reservoir of F. tularensis. Rodent species probably do not serve as true reservoir hosts of tularemia.