Update on rickettsioses in Slovakia.

The reported incidence of vector-borne diseases including various cases of Rickettsioses in humans is increasing due to a combination of climatic and social factors, escalating the opportunities for contact between people and ticks, fleas or lice. Many of the emerging infectious diseases currently challenging human health in Europe are transmitted by ticks which normally feed on domestic or wild animals. Each Rickettsia spp. has one or several tick vectors, and their geographical distribution varies according to geographical conditions; e.g.; altitude or temperature, which is gradually changing due to a global warming. Evidence of Rickettsia spp. particularly of a newly discovered species is a strong indication that a great number of diseases may be caused by so far undetected or unrecognized organisms. Their diagnosis relies mostly on rare "spot like" cooperation of clinicians with scientists, the members of the working groups that are devoted to the scientific studies of the corresponding research areas. The clinical picture of the disease caused by rickettsiae varies significantly from flu like symptoms to severe fatal outcomes, reflecting the various factors, e.g. a variability of virulence of rickettsial species due to cell invasion, dissemination of rickettsiae, genomics, immune response of an infected organism, or a tricky impact of a treatment. Several major reviews on rickettsioses have been previously published, e.g. in 1997 (Raoult and Roux, 1997a), in 2005 (Parola et al., 2005), and in 2011 (Botelho-Nevers and Raoult, 2011). In this work we intend to present a short historical overview and to describe new trends in research studies of rickettsiology. The main focus will be on rickettsioses affecting Europe΄s population.

Isolation of rickettsia helvetica from ticks in Slovakia.

UNLABELLED: To date, only three rickettsial species have been found in ticks in Slovakia by serological and/or molecular-biological techniques, namely Rickettsia slovaca, Candidatus rickettsia IRS, and Rickettsia raoultii. Recently, we succeeded in isolation of the forth species, Rickettsia helvetica from Ixodes ricinus, the most frequent tick in Slovakia. The isolation, positive for 10% of tested ticks, was performed on XTC cells by the shell-vial technique, Gimenez staining and light microscopy. The infected cell cultures contained rod-shaped particles morphologically identical to rickettsiae. The isolation was confirmed by direct detection of a fragment of the R. helvetica gene for citrate synthase in the positive ticks by PCR and its subsequent cloning, sequencing and comparison with the database. KEYWORDS: Rickettsia helvetica; isolation; Ixodes ricinus; Slovakia.

Ultrastructural study of the life cycle of Rickettsia slovaca, wild and standard type, cultivated in L929 and Vero cell lines.

Ultrastructural changes induced by Rickettsia slovaca standard type (ST) and wild type (WT) were examined during their life cycle in L929 and Vero cells. R. slovaca invaded the cytoplasm of the host cell by phagocytosis on the 1st d p.i. Rickettsiae adhering to the cytoplasmic membrane were engulfed by cellular extensions and occurred in phagocytic vacuoles. Binary fission of rickettsia was observed. The nuclear chromatin of eukaryotic cells was rearranged and condensed during 3rd and 6th d p.i. Finally, loss of the plasma membrane integrity, destruction of cytoplasm and nucleus resulted in cell lysis. Degeneration of the host cell caused by WT and ST was observed after 4 and 5 d p.i. in L929 cells and after 3 and 6 d p.i. in Vero cells, respectively. WT type was able to penetrate into the nucleus of the host cell and was responsible for dilatation of the perinuclear space and endoplasmic reticulum, causing more pronounced and different cytopathological changes than the ST.

Incidence of various tick-borne microorganisms in rodents and ticks of central Slovakia.

In this study, we detected Rickettsia helvetica, Candidatus Midichloria mitochondrii, Anaplasma phagocytophilum, Ehrlichia muris, Candidatus Neoehrlichia mikurensis, and Bartonella sp. infections in wild rodents and ticks collected from the vegetation of central Slovakia. The microorganisms were identified by PCR and sequencing. Yellow-necked mice (Apodemus flavicollis) were infected with E. muris and Bartonella sp., while ticks Ixodes ricinus collected from the vegetation were infected with R. helvetica, Candidatus M. mitochondrii, Candidatus N. mikurensis, A. phagocytophilum, and E. muris.

Rickettsial agents in Slovakian ticks (Acarina, Ixodidae) and their ability to grow in Vero and L929 cell lines.

A total of 80 adult ticks (55 Haemaphysalis inermis, 12 Dermacentor reticulatus, 11 D. marginatus, 2 Ixodes ricinus) were collected from vegetation in three areas of Slovakia (forest and pasture habitat) in central Europe. Forty-six (46 ticks) (57.5%) of all species tested were positive by the hemocyte test, PCR assays based on the gltA and ompA genes showed a Rickettsiaceae infection in 77.5% of the ticks, whereas only one H. inermis tick was positive for Anaplasmataceae on a 16S rRNA-based PCR. Isolation of rickettsiae was attempted on all collected ticks by means of the shell vial technique, 52 isolates of which were inoculated into Vero cells and 28 into L929 cells. Rickettsiae were detected in 50% (40/80) of the cell lines using the Gimenez staining method, whereas 33.8% (27/80) of the cell lines were PCR-positive for Rickettsia species. The presence of rickettsiae was shown by PCR to be around 30.8% (16/52) in Vero and 39.3% (11/28) in L929 cell lines. Sequencing results showed that detected infections were Rickettsia sp., R. raoultii, and Anaplasma phagocytophilum in ticks, and R. slovaca in cell lines. This is the first report of R. raoultii in Slovakia. Observations by electron microscopy of the R. slovaca isolate from Vero cell lines showed a microcapsular layer, typical Gram-negative cell wall, and a cytoplasmic membrane.

[Acute myocardial infarct as a result of external compression caused by an expanding pulmonary adenocarcinoma]. / Akutní infarkt myokardu jako dusledek zevní komprese prorustajícího adenokarcinomu plic.

The case report describes a rare cause of acute myocardial infarction. 68-years old man with the cancer triplicity (follicular and papillar carcinoma of the thyroid gland, Grawitz tumor, bronchogenic carcinoma with generalization) was admitted to the intensive care unit for the sudden onset of chest pain, positivity of cardioselective enzymes and signs of cardiogenic shock. Echocardiographic examination proved diaphragmatic akinesis with low left ventricular ejection fraction about 30%. The acute coronary angiogram revealed unimportant atherosclerotic narrowing of the left coronary artery and a long significant stenosis in the proximal portion of the right coronary artery untypical for atherosclerotic lesion suspicious of extramural compression. A pathological vascularization to the extramyocardial region was documented during right coronary angiogram. The patient was treated conservatively and finally transferred to the pulmonary disease department, where he died of progression of the cancer disease two months later. Postmortem examination found spreading of the bronchogenic adenocarcinoma to the pericardium along the course of the right coronary artery, but neither direct infiltration of the vessel wall by the tumor nor atherosclerotic disease of the proximal portion of the right coronary artery were proved. These findings together with the coronary angiogram demonstrate a rare cause of the myocardial infarction due to the extramural compression by the malignant tumor.

Comparison of virulence of Coxiella burnetii isolates from bovine milk and from ticks.

Laboratory animals (mice and guinea pigs) were infected with the isolates of Coxiella burnetii (Derrick, 1939) obtained from bovine milk (M18 and M35) and the ticks Ixodes ricinus (Linnaeus, 1758) and Dermacentor marginatus (Sulzer, 1776) (Kl3 and Kl6, respectively), and with the reference strain Nine Mile. Neither mortality nor lethality occurred with the mice. Antibody response in mice infected with isolates from milk was lower (1:16-512) than that from ticks (1:32-4096). Onset of seropositivity also occurred later - on the 10th day post-infection (p.i.) for M18 and M35 in comparison with the 7th day for Kl3 and Kl6. In guinea pigs, infection manifested by fever. The fever was less evident in guinea pigs infected with isolates from milk (39.5-40.1 degrees C) than in guinea pigs infected with isolates from ticks (39.5-40.6 degrees C). Partially engorged females of Dermacentor reticulatus (Fabricius, 1794) were inoculated with isolates M18 and Kl3. No differences in the multiplication of C. burnetii in haemocytes between these two isolates were ascertained.

Importance of surveillance of tularemia natural foci in the known endemic area of Central Europe, 1991-1997.

INTRODUCTION AND PURPOSE: Marked activation of natural foci of tularemia in the known endemic area of Central Europe, comprising the borderland of Slovakia, Austria and the Czech Republic, led to an epidemic outbreak in western Slovakia and an increase in the number of human tularemia cases in the adjoining regions of northeastern Austria and southern Moravia from 1995 to 1997. The aim of this paper was to present the results of a longitudinal study on the prevalence of infection with Francisella tularensis in small mammals and ticks in six localities of western Slovakia and northeastern Austria from 1991 to 1997, and to draw attention to the importance of systematic surveillance of natural foci for epidemiologic prognoses and for taking preventive measures. METHODS: In Slovakia, three localities of the Záhorie lowland were monitored for 3 to 6 years (1991 to 1996) for the presence of F. tularensis in small terrestrial mammals. In Austria, small mammals and ticks from three localities were investigated for 1 to 3 years (1994 to 1997). Spleens of live-trapped animals (at 1- to 2-month intervals) and ticks collected by flagging (in 6- to 8-week intervals) were examined by inoculation of pools into white mice, or individually by cultivation. RESULTS: In four localities under investigation (three localities in western Slovakia and one in Austria), a nearly simultaneous flare-up of tularemia epizootics was recorded in the autumn of 1994. The highest mean prevalence of infection in small mammals was 3.9% in the last quarter of the year, which along with isolations of F. tularensis from Dermacentor reticulatus ticks collected from vegetation in the locality of Austria (1.3% positivity), indicated the high epizootic activity of foci. F. tularensis was isolated from five rodent species--Apodemus flavicollis, A. sylvaticus, Clethrionomys glareolus, Microtus arvalis and Sorex araneus. From 1995 to 1996, the persistent activity of the focus was repeatedly confirmed by isolations of F. tularensis from D. reticulatus ticks. In 1995 a natural focus of tularemia was detected in a further locality of Austria, where the agent was isolated from D. reticulatus. The increased epizootic activity of foci in the endemic region of Central Europe indicated a higher risk of acquiring tularemia for humans and was, in fact, followed by an epidemic outbreak in western Slovakia as well as by a large number of cases in Austria and the Czech Republic. CONCLUSION: Our findings clearly demonstrate that long-term surveillance of natural foci in endemic regions provides useful information on the activation of tularemia foci before the onset of epidemic outbreaks, thus permitting timely epidemiologic prognoses and the institution of preventive measures.

Genetic diversity of Borrelia burgdorferi sensu lato isolates obtained from Ixodes ricinus ticks collected in Slovakia.

In Europe, Borrelia burgdorferi sensu lato is diverse, including B. burgdorferi s.s., B. garinii, B. afzelii, B. valaisiana and B. lusitaniae. In this study, we focused on the distribution of the different B. burgdorferi species among Ixodes ricinus adult ticks collected in an endemic area within Slovakia. We compared results of prevalence of B. burgdorferi infection in ticks obtained by immunofluorescence (IF) and by isolation. Isolates were characterized by restriction fragment length polymorphism (RFLP) of the rrf-rrl intergenic spacer genes using MseI. Using immunofluorescence we observed that 56/114 (49%) ticks were infected by B. burgdorferi s.l. Males were found to be more often infected (32/57, 56%) than females (24/57, 42%) but the difference was not significant (p = 0.1895). From the same 114 ticks a total of 37 isolates were obtained: 19 from males (33%) and 18 from females (32%). The RFLP identification revealed 25 B. afzelii (68%), 5 B. garinii (14%), 5 B. valaisiana (14%) and 2 B. lusitaniae (5%). The infection in ticks was more often detected by IF than by isolation (p = 0.0153) and isolation success was higher when the infection degree in ticks was high (p = 0.0397). The infection prevalence observed in this area is among the highest observed in Europe.

[Contamination in areas of Slovakia with strains of Coxiella burnetii imported from abroad]. / Problém kontaminovania niektorých oblastí Slovenska kmenmi Coxiella burnetii zavlecenými zo zahranicia.

Following the upsurge of Q fever epidemic in the village Jedl'ové Kostol'any, Nitra district, Slovakia, in April 1993, the possibility of the penetration of coxiellae into free nature was investigated for the next two years. Micromammals and their ectoparasites, primarily ticks, were employed as indicators of the presence of the infection in nature. The infestation of mammals and their ectoparasites was followed in J. Kostol'any on the farm where the goats were housed as well as in its close proximity. The infestation of ticks with coxiellae was further followed in four localities of the Rimavská Sobota district, where the goats after the eradication of Q fever outbreak in J. Kostol'any were transferred for breeding. The presence of coxiellae was not proved in these investigated localities. It is suggested that the pathogenicity of imported C. burnetii strains through Bulgarian goats was decreased under local environmental conditions including the ability to circulate freely in nature or these strains found inconvenient conditions for maintaining their viability.

Prevalence of ticks infected with Francisella tularensis in natural foci of tularemia in western Slovakia.

The prevalence of ticks infected with F. tularensis was followed during a systematic surveillance in endemic area of tularemia in western Slovakia over the years 1984-93. Ticks were collected from vegetation in localities of Podunajské Biskupice, in the vicinity of the capital of Slovakia, Bratislava, near the river Danube. In total 6033 ticks, mostly adults of Dermacentor reticulatus and Ixodes ricinus (4994 and 1004, respectively) and 35 nymphs of Haemaphysalis concinna, were examined for the presence of F. tularensis. Out of 4542 starving ticks, 34 F. tularensis strains were isolated predominantly from D. reticulatus (30), and to a smaller extent also from I. ricinus (3) and H. concinna (1). Natural infection with F. tularensis was further proved from 1491 adults of D. reticulatus fed on laboratory animals, rabbits and white mice, together in 27 cases. From that, 21 times it was by positive isolation either from suspensions of partly or fully engorged ticks and their feaces, or from spleens of animals dead after the feeding of ticks. In addition, solely the development of antibodies against the agent was confirmed in 6 rabbit hosts. The presence of F. tularensis in all the above mentioned tick species and namely the relatively high and permanent infestation of D. reticulatus adults, ranging between 0.5-2% during the followed time period, demonstrated the maintenance of active natural focus of tularemia in the area under study. The present paper also emphasizes the epidemiologic consequence of various species of ticks in endemic foci of tularemia and the aspect of possible ways of transmission of the agent to humans.

Isolation of Coxiella burnetii and of an unknown rickettsial organism from Ixodes ricinus ticks collected in Austria.

Two strains of Coxiella burnetii and two strains of an unidentified rickettsial organism were isolated for the first time from Ixodes ricinus ticks collected in the Alpine region of Tirol, Austria. The C. burnetii strains belong to the group of agents causing acute forms of Q fever. The other two strains of isolated rickettsial agent share some antigenic epitopes with C. burnetii and R. prowazekii but they differ from them by their high sensitivity to freezing and refreezing and by poor multiplication in yolk sacs of chick embryos. There is at present no evidence that these organisms cause human illness and no ecological information is available. We suggest they may be some new species of rickettsiae or rickettsia-like organisms.

Use of partially engorged female ticks as laboratory animals in microbiological research.

Partially engorged female ticks were used as laboratory animals in microbiological research. The ticks, which were inoculated intracoelomally, became a convenient substrate for the detection of viruses, rickettsiae and protozoal parasites. This research concerned the isolation of newly recovered micro-organisms, the study of development, structure and distribution of microbial agents in ticks, and the study of their interaction with other pathogens or symbionts during mixed infection in a tick body. The isolation and maintenance of Rickettsiella phytoseiuli, the organism not of tick-borne origin, was achieved. For use in Central Europe the tick Dermacentor reticulatus is recommended for the above investigations.

Immunochemical and antigenic characterization of Coxiella burnetii strains isolated in Europe and Mongolia.

SDS-PAGE, immunoblotting and serological methods such as microimmunofluorescence (MIF) test and ELISA were used to compare protein and lipopolysaccharide (LPS) profiles and antigenicity of 12 Coxiella burnetii strains isolated mostly from ticks in Europe and Mongolia with three reference C. burnetii strains originating from USA, namely Nine Mile from tick, Priscilla from goat placenta and S from human heart valve. Among strains from Europe and Mongolia, no significant differences in protein and LPS profiles were observed, irrespective of their origin, i.e. the country and source of isolation. The LPS profiles of these strains appeared to be more related to those of Nine Mile strain associated with acute Q fever, than to those of strains S and Priscilla associated with chronic Q fever. In immunoblots all strains isolated from Slovakia and Poland reacted more expressively with rabbit serum against Nine Mile than with serum against Priscilla strain. In the MIF test and ELISA there were no substantial differences in antibody-binding capacity between the reference and newly isolated C. burnetii strains, except for strain Priscilla reacting with homologous serum in lower antigenic concentration than other strains under study. However, in the MIF test much higher antigenic concentrations of each C. burnetii strain was required to detect antibodies in the Priscilla serum than in the Nine Mile, Luga and S sera.

[The ecology index, an epidemiologic marker of foci of Lyme borreliosis]. / Ekologický index, epidemiologický marker ohnísk lymskej boreliózy.

The ecological index (EI) as defined by Schulze et al. was determined in three localities of western Slovakia (Malacky, Podunajské Biskupice, Olichov) using the following criteria: suitability of the habitats for the occurrence of Ixodes ricinus ticks and their number, accessibility of habitat for men (I. phase); furthermore the abundance and infection rate of ticks (II. phase). A scoring procedure (point values of 1-5 for each parameter) was used to evaluate the EI of three localities studied. Based on obtained values it is suggested to differentiate foci as those with a definite (Malacky), potential (Podunajské Biskupice, Olichov), minimal and unprobable risk of transmission. The EI is considered a helpful method for assessing areas of risk for the transmission of Lyme borreliosis, indicating also the necessity of appropriate preventive measures.

The prevalence of small terrestrial mammals infected with tick-borne encephalitis virus and leptospirae in the foothills of the southern Bavarian forest, Germany.

In the district of Grafenau/Freyung (Bavaria, Germany), 266 specimens of small terrestrial mammals of 8 species were captured using live traps. From these mammals, Apodemus flavicollis (42.1%) and Clethrionomys glareolus (39.5%) were prevalent. All animals were tested for neutralizing antibodies to tick-borne encephalitis (TBE) virus and agglutinating antibodies to leptospirae. Seropositivity against TBE virus was 14.0% and against leptospirae 7.9%, respectively. Seropositivity to leptospirae appeared to be primarily to Leptospira grippotyphosa, less to Australis and occasionally to Javanica serovars. Only one A. flavicollis specimen was positive to both pathogens tested. The parasitocoenosis of trapped micromammals with ectoparasites consisted of 69.5% from ticks (mainly Ixodes ricinus, less from I. trianguliceps), 16.1% of mites (primarily Laelaps agilis) and 14.3% of fleas (mostly Ctenophthalmus agyrtes).