The influence of meteorological factors on the risk of tick-borne encephalitis infection.

OBJECTIVES: The aim of this work was to analyze the relationship between new cases of clinical tick-borne encephalitis (TBE) and various meteorological and seasonal predictors. MATERIAL AND METHODS: The modelling is based on national data from the Czech Republic for the period 2001-2016 in daily resolution, namely on average temperatures, average relative air humidity and the number of TBE cases classified according to the date of the first symptoms. Four variants of a negative binomial model from the generalized additive model class are used. The basic model relates the occurrence of TBE to the lagged ambient daily average temperature and daily average relative air humidity and their interaction with the lag reflecting the incubation period and other factors. The lag value was estimated via the optimization procedure based on Akaike information criterion. The model also includes the effect of the season and the effect of the day of the week. To increase the biological plausibility, the basic model has been expanded to account for possible time-varying effects of meteorological variables and to incorporate multiple lags. RESULTS: The most statistically significant effect is the within-year seasonality and then the interaction of the temperature and relative air humidity. The relationship of both meteorological factors and their interactions vary throughout the activities season of the hostquesting Ixodes ricinus. This also changes the conditions of occurrence of the new clinical cases of TBE. The time-varying effect of meteorological factors on the incidence of TBE shows non-trivial changes within a year. In the period before the middle of the calendar year (around the week 22) the effect decreases, then it is followed by an increase until the week 35. CONCLUSION: Flexible models were developed with quantitatively characterized effects of temperature, air humidity and their interaction, with the delay of the effect estimated through the optimization process. Performance of the model with multiple lags was checked using independent data to verify the possibility of using the results to improve the prediction of the risk of clinical cases of TBE uprise.

The occurrence of Ixodes ricinus ticks and important tick-borne pathogens in areas with high tick-borne encephalitis prevalence in different altitudinal levels of the Czech Republic Part II. Ixodes ricinus ticks and genospecies of Borrelia burgdorferi sensu lato complex.

STUDY OBJECTIVE: Three years long research study (2011-2013) on population density of Ixodes ricinus and the infection rate of the pathogens that they transmit was conducted in four topographically distant areas in the Czech Republic. In the previous decade (2001-2010) thirteen loci with increased incidence of tick borne encephalitis cases were defined, suggesting the permanent interaction of human population with ticks and indicating the landmarks for study of the presence of other tick borne pathogens. The work program included the identification of existing spectrum of spirochetes from Borrelia burgdorferi sensu lato complex and the conditions of their occurrence and distribution. MATERIAL AND METHODS: In the areas of the Ústí nad Labem Region, Olomouc Region, South Bohemian Region, and Highlands Region, 600 m2 plots were selected in the local optimal I. ricinus habitats where tick flagging was performed every year in the spring-summer and autumn seasons of the tick questing activity. Collected adult ticks (1369 males and 1404 females) were individually screened for B. burgdorferi s. l. spirochets. RESULTS: Spirochetes from B. burgdorferi s.l. complex were detected in all 13 studies sites in all altitudes from 280 to 1030 meters a. s. l. The total rate of infection was determined as 11.4% (males 10.4%, females 12.4%) with range limits from 1.4% (Ústí nad Labem in 2011) to 19.7% (South Bohemian Region, 2012).Genospecies were detected in various proportions and in different combinations: Borrelia afzelii, B. garinii, B. burgdorferi s. s., B. bavariensis, B. bissettii, B. valaisiana, B. spielmanii and B. lusitaniae. The three-year observation justifies the assumption that the regional differences in infectivity of I. ricinus are based on the character of the local biocenosis of the respective region. The dynamics of its seasonal changes, conditioned by climatic factors, determines the annual differences. CONCLUSION: Three of the medically most important Borrelia species formed a core group among all detected genospecies. B. afzelii was a dominated one (115 detections), followed by B. garinii (100) and by B. burgdorferi s.s. (19). Other genospecies were detected sporadically. However, the detection of B. bissettii should be emphasized due to the recently proven pathogenic effects of this genospecies and yet little-known sporadic expansion in the Czech Republic. The medical importance and distribution of other sporadically occurred genospecies is also discussed.Key words: Ixodes ricinus - Borrelia afzelii - B. garinii - B. burgdorferi s. s. - B. bavariensis - B. valaisiana - B. spielmanii - B. lusitaniae - B. bissettii - distribution - altitude - season - medical importance.

The occurrence of Ixodes ricinus ticks and important tick-borne pathogens in areas with high tick-borne encephalitis prevalence in different altitudinal levels of the Czech Republic Part I. Ixodes ricinus ticks and tick-borne encephalitis virus.

STUDY OBJECTIVE: The aim of the three-year study (2011-2013) was to monitor population density of Ixodes ricinus ticks and its infection rate with the tick-borne encephalitis virus in areas with a high incidence of tick-borne encephalitis as reported in the previous decade 2001-2010. Such a comprehensive and long-term study based on existing epidemiolo-gical findings has not previously been conducted in Europe. MATERIAL AND METHODS: In the areas of the Ústí nad Labem Region, Olomouc Region, South Bohemian Region, and Highlands Region, 600 m2 plots were selected in the local optimal I. ricinus habitats where tick flagging was performed every year in the spring-summer and autumn seasons of the questing activity. In total, 18,721 I. ricinus ticks (1448 females, 1425 males, and 15,848 nymphs) were collected and investigated. RESULTS AND CONCLUSION: The results have shown that the differences in the infection rate of I. ricinus observed between regions are driven by variation in the density of the local I. ricinus populations which is influenced by the characteris-tics of the whole local biocenosis. The overall prevalence estimate of TBE virus in Ixodes ricinus ticks at the altitudes below 600 m a.s.l. was 0.096 % (95% CI 0.055-0.156) for nymphs, and 0.477 % (95% CI 0.272-0.773) for adults. The dynamics of the seasonal variation in I. ricinus populations, depending primarily on the climatic factors, are behind the interyear differences in the infection rate of ticks and, consequently, in the epidemiological situation of tick-borne encephalitis. The nymph to adult ratio was 5.5 on average but showed great interregional variability (from 10.3 in the Ústí nad Labem Region to 1.8 in the Highlands Region). It might be used in the future as one of the indicators of the composition of the local I. ricinus population and of the level of the circulation of tick-borne pathogens in zoonotic sphere and also for use in the health risk assessment in a given area. Despite the permanent expansion of ticks and tick-borne pathogens in higher altitudes the high risk limit for human infection with tick-borne encephalitis is 600 m a.s.l. in the Czech Republic.

Sixty years of research of tick-borne encephalitis--a basis of the current knowledge of the epidemiological situation in Central Europe.

Tick-borne encephalitis (TBE) virus was isolated for the first time in Central Europe in 1948 from both a patient and Ixodes ricinus ticks collected in the area where the patient had been tick bitten (the Beroun area - Central Bohemia) and concomitantly from a TBE patient in Moravia (the Vyskov area). Another priority discovery was alimentary transmission of TBE virus via the milk from tick infected grazing goats that was made during a TBE outbreak in Roznava (SE Slovakia). This outbreak of 660 cases has been the largest of its kind. Both of these discoveries were a challenge to multidisciplinary research into the natural focality of TBE. The results obtained were published by Czech and Slovak authors in the first European TBE monograph (1954) and were the stimulus for further research in this area. From the epidemiological point of view, among others, the impact of meteorological factors (on TBE incidence associated with I. ricinus host-seeking activity) and recreational nature of TBE were clearly defined then. At the same time, TBE became a notifiable disease (since 1971 laboratory confirmed TBE cases only). In the following decades, the phenomenon of natural focality of TBE (including anthropic impacts) was extensively studied and the determinants of high-risk areas in the field were analyzed. The results were used in the creation of I. ricinus and TBE risk prediction maps for the Czech Republic generated for the first time in Europe using LANDSAT 5 satellite data and GIS technology (1990). In the early 1990s (in particular since 1993), similarly to other countries, the Czech Republic reported a sharp rise in TBE cases that continues, with some fluctuations, until now. The cooperation with climatologists in the analysis of historical data, current epidemiological observations, and study of I. ricinus in the field have shown a decisive impact of the ongoing climate change. The analysis of the socio-economic conditions in high-risk areas for TBE has not revealed any impact of these conditions on TBE morbidity. The recreational factor that is influenced by the weather changes has a considerable impact. The seasonal trend of TBE cases shows large fluctuations as were seen in 2006, 2009, and 2010, also as a result of weather changes with seasons. This clearly implies the need for using long time series of data, covering at least a decade, to be able to draw general conclusions as is the case in the present study (2001-2010). The data broken down by Administrative Region display substantial interregional differences. Of 14 Administrative Regions of the Czech Republic, three exhibits a linear trend in TBE incidence, with a minimum deviation from the baseline, four Regions show different downward linear trends, but seven Regions display different upward trends. The upward trend is most obvious in the Highlands (Vysocina) Region where it is associated with the prevalent orographic conditions and increase in the incidence of I. ricinus ticks at higher altitudes. The knowledge of the area where the patient was tick bitten that is entered in the Epidat database as the "probable area of TBE infection acquisition" is helpful in identifying high-risk areas for TBE. By matching the area of TBE acquisition with that of the patient's domicile we revealed that TBE patients had to travel to areas other than their area of domicile to acquire TBE and thus also significance of the areas of TBE acquisition at the country level. The population of the Prague Region (NUTS3 CZ010) can be used as an illustrative example, with 37.7% of TBE cases only reported to be acquired in the Prague Region while 33.4% of TBE cases were associated with travel to the Central Bohemian Region and 13.9% of TBE cases were imported from the South Bohemian Region (the rates of TBE cases imported from other Regions were less than 5%). And conversely, the residents of the South Bohemian Region (CZ031), with the highest number of TBE cases ever in the Czech Republic, acquired TBE in the region of domicile at a rate of 99.5%. These rates are clearly associated with the recreational potential of various Regions. The probable area of TBE acquisition is identified by cadastral community. In the light of the natural focality of TBE, the analysis of the local environmental factors involved in the circulation of TBE virus in the wild environment is required to determine the high-risk areas and local risk level. Although outbreaks of TBE cases in humans are indicative of TBE natural foci, more data is needed to delineate such areas. And similarly, the absence of TBE cases in humans over a period of time may not be indicative of a no risk area.

Changes of meteorological factors and tick-borne encephalitis incidence in the Czech Republic.

OBJECTIVES: The primary objective was to analyze the influence of short-term meteorological changes during the vegetation period on the incidence of tick-borne encephalitis (TBE) in the 1990s, characterized by a dramatic increase in reported TBE cases in the Czech Republic and other European countries. Furthermore, the relationship between TBE incidence and meteorological conditions in the previous winter season was studied. MATERIAL AND METHODS: The TBE incidence data were acquired from the EPIDAT database of the National Institute of Public Health (NIPH). Analyzed were a total of 4637 cases reported in Bohemia (1994-2004). Meteorological data were from the database of the Czech Hydrometeorological Institute in Prague and originated from 22 meteorological stations located in high TBE incidence areas in Bohemia. RESULTS: A linear relationship was found between TBE incidence and temperature factors in all the years under study. Lagged cross-correlation analysis (with the time lag corresponding to the incubation period from the infected tick bite to the onset of TBE symptoms) revealed a close correlation between TBE incidence and weekly mean air temperature with a lag of 1 to 5 weeks. When considering the previous winter period, the closest relationship was found between TBE incidence and the previous-winter frost index, followed by the minimum air temperature. CONCLUSION: A review is presented of the effects of the currently observed climate change on TBE incidence as compared with the data reported in the 1950s. Results of parallel analyses of other factors potentially implicated in higher TBE incidence in the 1990s lead to a critical rejection of the conclusion previously drawn by some authors that the collapse of communism and subsequent dramatic socio-economic changes might have a decisive influence on TBE incidence in Central Europe. The rise in TBE cases reported in West European countries where no such political changes took place confirms the refutation.

Correlation between meteorological factors and tick-borne encephalitis incidence in the Czech Republic.

The objective was to discover whether there any demonstrable relationships exist between the incidence of human tick-borne encephalitis (TBE) cases and current meteorological factors and to specify their character. Epidemiological data were extracted from the EPIDAT database (National Institute of Public Health, Prague). Analyzed were a total of 4,613 TBE cases registered in the whole Czech Republic (1994-2001) and 4,637 cases registered in the territory of Bohemia (1994-2004). Meteorological data were from the database of the Czech Hydrometeorological Institute in Prague. A linear relationship has been found between TBE incidence and temperature factors in all the years under study. Lagged cross correlation was used (with the lags in time respective to incubation period from infected tick attack to initial TBE symptoms) and close relations were found for daily mean air temperature and lags 6-14 days (with a peak of 9 days). Effects of the current course of the meteorological situation (as well as long-term year-to-year changes) on TBE incidence are, foremost, mediated by the influence of climatic factors on Ixodes ricinus ticks and their host-seeking activity; under certain conditions, also by the effects of momentary weather on human behavior as TBE in the Czech Republic is a recreational disease connected with outdoor activities.

Altitudinal distribution limit of the tick Ixodes ricinus shifted considerably towards higher altitudes in central Europe: results of three years monitoring in the Krkonose Mts. (Czech Republic).

The aim of the study was to monitor present status of the Ixodes ricinus tick vertical distribution in the Krkonose Mts. (Czech Republic) and evaluate its potential changes. Two methods were used: monitoring of tick presence on dogs in 2001-2002 and direct monitoring of host-seeking ticks by flagging on two vertical transects reaching from 620 to 1270 m above sea level (a. s. l.) and from 600 to 1020 m in 2002-2003. Moreover ticks were monitored by flagging at another 13 localities situated in 800-1299 m a. s. l. in 2003. Both monitoring methods revealed an obvious shift in altitudinal distribution limit of the tick I. ricinus in the Krkonose Mts. They both showed that ticks recently penetrate even up to a timberline (approximately 1250 m a. s. l.). The number of recorded host-seeking ticks rapidly decreased with increasing altitude. Whereas the average number of recorded nymphs converted per 60 minutes of flagging reached 15.2-25.7 nymphs in 700-799 m a. s. l. and 3.3-23.3 in 800-899 m, it was 4-9.6 nymphs in 900-999 m, 1.5-1.7 nymphs in 1000-1099 m and only 0.2 nymph in 1100-1299 m a. s. l. The observed shift of the tick altitudinal distribution limit at the same time results in extension of areas with potential risk of tick-borne diseases.

Prevalence of Borrelia burgdorferi sensu lato genospecies in host-seeking Ixodes ricinus ticks in selected South Bohemian locations (Czech Republic).

In selected localities of Ceské Budejovice and Ceský, Krumlov districts, well known by stable high incidence of tick-borne encephalitis (TBE) human cases but with low incidence of Lyme borreliosis, monitoring of Borrelia burgdorferi sensu lato (s.l.) in Ixodes ricinus ticks was performed. Research was also aimed at the spread of I. ricinus to mountain areas of this region (National Park Sumava), as well as at investigating this tick for B. burgdorferi s.l. genospecies and TBE virus infection. Altogether 498 nymphs, 88 females and 11 males of I. ricinus from lower locations and 58 nymphs from mountain locations (760-1080 m above sea level) were tested by polymerase chain reaction. In lower locations total prevalence of Borrelia burgdorferi s.l. in Ixodes ricinus ticks was 35%. Single infection of Borrelia afzelii, B. garinii and B. burgdorferi sensu stricto (s.s.) was found in 59, 50 and 63 ticks, respectively (i.e. in 12.8, 11.2 and 14.1%). Double infection was found in 42 ticks (6.0%) and triple infection in three ticks (0.4%). The high frequency of B. burgdorferi s.s. exceeds the as yet reported occurrence in Central Europe. These circumstances are discussed. In mountain locations B. afzelii was found in five ticks, that including two co-infection with B. garinii, in elevations of 762 m and 1024 m above sea level, respectively. This fact signals a real danger of human infections in a region that was previously deemed to be without risk. Moreover, this region is more and more the target destination of tourist activities. The results also suggest that the penetration of infection can be rapid and formation and establishment of natural focus of Lyme borreliosis might be rather quick.

Socio-economic conditions and other anthropogenic factors influencing tick-borne encephalitis incidence in the Czech Republic.

Laboratory confirmed cases of tick-borne encephalitis (TBE) have been reported in the Czech Republic since 1971. Peaks of incidence occurred at 2 to 5-year intervals. The incidence in the eighties was generally lower than in seventies. In the nineties there was a sharp increase in incidence that peaked in 1995 (7.2/100,000). TBE cases occur from April to November with maximum incidence in July. During the last decade the second peak of incidence occurred in most years in September and/or October. In the last decade, an extension of the TBE season towards the spring and autumn periods has been observed. In addition to a general increase in TBE incidence in the last decade, some other changes in the epidemiology of TBE were observed in the Czech Republic. During the whole period sex-specific incidence was higher in men than in women (men to women ratio 1.5:1). Age-specific incidence over recent years increased steadily in children and adolescent age groups. In the ten-year age groups from 25 to 65, it remains practically at the same level (6-8/100,000). In older persons it fell to 2-3/100,000. Tick bites infect patients mainly during their recreational activities. A very small proportion (less than 1%) acquires the infection through the alimentary route. We have tested the hypothesis whether the increase in TBE incidence in the nineties was due to economical or social changes after the velvet revolution of 1989. Between 1991 and 1995 unemployment largely remained on the same level (between 2-3%). Over the next years the percentage of unemployed persons increased rapidly to 9.3% in 1999 (7.8% in 2001). This trend differs significantly from the trend of TBE incidence that peaked in 1995. No correlation between the district incidence of TBE and the district percentage of unemployment in the years 1997-2001 was found (r=-0.20). The percentage of unemployed persons among the TBE cases was 1-3% in contrast to the Czech Republic figures which were 5-9% for the same period. The gross domestic product in USD per capita increased from dollars 2,600 in 1991 to dollars 5,000 in 1995. Since then it has varied between dollars 4,800 and dollars 5,600. This trend, therefore, differs from the trend of TBE incidence as well. Among the TBE cases the percentage of foresters and other persons working in the forests in the years 1997-2001 was 0.5-1%. The behavioral and socio-economic aspects of TBE cases remained stable despite the political changes which have take place in the Czech Republic since the beginning of the nineties. They are not, therefore, responsible for the increased TBE incidence. In the industrial areas most polluted by SO2 in the past in northern Bohemia the sharp increase in TBE incidence seems to be connected with measures aiming to eliminate SO2 emissions from brown coal power stations.

[Effects of climate change on the incidence of tick-borne encephalitis in the Czech Republic in the past two decades]. / Vliv zmen klimatu na výskyt klíst'ové encefalitidy v ceské republice v uplynulých dvaceti letech.

The study objective was an attempt to explain uneven distribution of the incidence of tick-borne encephalitis (TBE) throughout the past two decades. A sharp rise was recorded in the last decade: while 2596 TBE cases were reported in the decade 1983-1992, as many as 5892 TBE cases were reported in the decade 1993-2002, with high rates persisting also in the following years (606 TBE cases reported in 2003). Data from the database TBE EPIDAT of the National Institute of Public Health, Prague, and that of the Communicable Diseases Information System, Ostrava, were used for analysis. Meteorological data were taken from the database of the Czech Institute of Hydrometeorology, Prague. In 1971 to 2003, 13 231 laboratory confirmed TBE cases were reported in the Czech Republic. Uneven distribution of these TBE cases in time is consistent with the observations of climate variation made between 1931 and 2000. The increase in the TBE incidence in the Czech Republic in the last decade was characterized by the following features: 1) higher rates of TBE cases manifested in regular TBE natural focuses, 2) reemergence of TBE in the same localities after 20 and more year intervals and 3) emergence of TBE in localities where it was not reported before. At the same time shifts in TBE seasonal trends (i.e. to March and October-November) were observed, associated with a TBE incidence peak in autumn. Field research revealed that the major factor are the climate-related changes in ecology of the TBE vector Ixodes ricinus and resulting variation in its population density. TBE emergence in new areas is linked to the occurrence of ticks Ixodes ricinus at higher altitudes (previously found at 700 m and currently spreading to 1200 m) as well as to a coming warm climate era.

Tick-borne encephalitis virus prevalence in Ixodes ricinus ticks collected in high risk habitats of the south-Bohemian region of the Czech Republic.

A survey for tick-borne encephalitis virus in trades ricinus ticks was conducted in May 2000 in two districts of the South-Bohemian region of the Czech Republic with a high occurrence of tick-borne encephalitis (TBE) in humans. Homogenized ticks were tested on PS cells, which were examined for any cytopathic effect, plaque assay and indirect fluorescent antibody test (IFT). The IFT has proved to be the most sensitive and most rapid test to demonstrate the virus in ticks. TBE virus was found in 17 of 187 pooled samples. consisting of a total of 2,968 ticks. The mean minimum infection rate was 0.6% for all tick stages combined. Infection rates in nymphs collected in different locations varied between 0.2 and 1.3% and between 5.9 and 11.1% in adult ticks. The observed TBE prevalence in ticks was compared with data obtained elsewhere in the Czech Republic. It is concluded that screening of ticks for TBE virus prevalence using IFT is a valuable indicator for the degree of risk to contract TBE in as particular habitat.

Cytotoxicity of cyclophosphamide, paclitaxel, and docetaxel for tumor cell lines in vitro: effects of concentration, time and cytochrome P450-catalyzed metabolism.

Cytotoxic effects of cyclophosphamide (CPA), paclitaxel (PCT), and docetaxel (DTX) and their modulation by cytochrome P450 (CYP) metabolism were studied by incubating cell lines L929 and P388D1 with or without rat liver microsomes. The microsomes themselves were not cytotoxic. P388D1 cells were more sensitive to CPA, PCT, and DTX than L929 cells. CYP2B1-, CYP3A-, and CYP2E1-induced microsomes effectively oxidized the prodrug CPA to cytotoxic products in 2-h incubation periods. Cytotoxicity of DTX and PCT for P388D1 cells became apparent 24 h after a 2-h incubation period with the drugs, and their effects were enhanced by CYP2E1 microsomes, but markedly decreased by CYP3A-induced microsomes. DTX and PCT without microsomes caused a dose-related cytotoxicity in P388D1 and HeLa cells. P388D1 and HeLa cells did not grow after a 24-h exposure to 1-10 microM DTX, but about 0.1% of cells survived exposure to 1-10 microM PCT. After 4 weeks of multiplication, the surviving P388D1 cells displayed lower sensitivity to DTX and PCT, but cytotoxicity in HeLa cells was unchanged and their growth ability decreased. In P388D1 cells, PCT with DTX (0.1, 0.5. 1, 2.5, or 5 microM) showed only additive cytotoxicity, although they reportedly act in different phases of the cell cycle. In P388D1 cells treated with DTX or PCT, normal mononuclear cells disappeared and the cell diameter increased up to threefold. Mulberry-like nuclei developed, giving rise to multiple nuclei, which were hyper- or hypochromatic. Chromatin condensation in some multiple nuclei and cell shrinkage of some cells fit the definition of apoptosis, but enlargement of the surviving cells and numerous hypochromatic nuclei do not. In conclusion, L929 and P388D1 cells incubated with microsomes enabled the role of various CYP enzymes in the effect of anticancer drugs to be assessed. The delayed cytotoxicity of DTX and PCT compared to that of CPA was related to their different mode of action. Fluorescent microscopy revealed quantitatively different effects of PCT and DTX on the nuclei, indicating that their mode of action may not be completely identical.

Electron microscopic demonstration of the interaction of liposomes and cells in vitro.

We have investigated the interaction of liposomes with the continuous cell lines P388D1 and L-132 and mouse peritoneal macrophages. To distinguish the liposomes from other vesicular structures, we have used liposomes with incorporated protein G and gold. A heterogeneous mixture of multilamellar liposomes 30 nm up to 1000 nm in size has been employed. Samples were examined at different time intervals. We found differences in the uptake of liposomes with regard to size and rate. Cells of a P388D1 monolayer took up liposomes by endocytosis very early after addition of liposomes and the number of lysosomes in their cytoplasm increased. In L-132 cells, first a fusion occurred between liposomes and the cell cytoplasmic protrusions, in the cytoplasm of which the mitochondria had multiplied. Peritoneal macrophages phagocytosed mainly large multilamellar liposomes and the membranous system of Golgi apparatus was the most prominent structure in the cytoplasm. Phagocytosis in P388D1 and L-132 cells was noted sporadically as late as 24 h after addition of liposomes to the cells.

Possible role of rainfall in the epidemiology of tick-borne encephalitis.

The assumption that the amount of rainfall in the summer months influences the frequency of TBE is based on the fact that the determining factor for the existence, development and activity of Ixodes ricinus is relative air humidity (RH) and the finding that low RH negatively influences dynamics of infection and infection rate of I. ricinus with TBE virus. A higher incidence of TBE then develops due to the coincidence of increased activity of ticks and recreational activity of humans after an intense prolonged rainfall. This presumption was verified by comparing the dynamics of rainfall as an indicator of RH in the tick environment with concomitant seasonal fluctuations of TBE incidence in humans. The total monthly rainfall in two regions of the Czech Republic was compared with data on the incidence of TBE in each respective region and period. Furthermore, in the district of Ceské Budejovice, typical by high prevalence of this disease, a detailed follow-up was carried out by decades. The comparisons were made between April and October from 1993 to 1995. The values of both variables were related to their long-term averages estimated within each particular period and region. Monthly data were analyzed for the Central Bohemian Region and the South Bohemian Region, both with a high incidence of TBE. The July above-average incidence of TBE was preceded by an above-average rainfall in June. If there was a below-average rainfall, there was a below-average incidence of TBE afterwards. Whenever, there was an above-average rainfall in August then there was an above average incidence of TBE in September, in the opposite case, no September peak of TBE occurred. The follow-up at ten-day intervals shows that any above-average rainfall was followed by an increased incidence of TBE, in every other ten-day period or two. This time shift is due to the incubation period, and partly also to the scattered attacks by ticks during the period of increased RH. The relationship found could be useful for the short-term prognosis of TBE incidence as well as for individual prevention of this serious infection by taking special care when going outdoors after heavy rain.

New serotype candidate of Neisseria meningitidis.

In spite of a large collection of MAbs used for the whole-cell ELISA (WCE) in the National Reference Laboratory for Meningococcal Infections in Prague 50-80% of N.meningitidis strains isolated in the Czech Republic remained non-typable (NT) and/or non-subtypable (NST). A project focused on the problem of NT/NST N.meningitidis was started and the new serotype candidate designated "22" resulted from this research. This paper presents the method of preparing and testing of the monoclonal antibody (MAb) specific for this new serotype and the first experience acquired from using it. The new serotype-specific MAb is of IgG3 class, does not react with any serotype/subtype reference strains and reacts in WCE with the strain used for its production and with some other NT/NST strains in the dilution 1:1,000. A collection of 97 N.meningitidis B:NT strains isolated from cerebrospinal fluid and/or blood of patients with invasive disease in the Czech Republic since 1973 to 1995 was serotyped using the new serotype "22"-specific MAb and 37 of these strains (38.2%) gave positive WCE result. The total number of 59 N.meningitidis B:NT strains isolated in 1995 from various clinical situations were serotyped and 26 of them (44.1%) were positive with the new serotype "22"-specific MAb. Seven of these N.meningitidis B:NT strains isolated in 1995 from various clinical situations were serotyped and 26 of them (44.1%) were positive with the new serotype "22"-specific MAb. Seven of these N.meningitidis B: "22" strains were isolated from cerebrospinal fluid or blood of patients with invasive meningococcal disease and prevailed in the age group of 0-4 years (5 cases). The significance of the new serotype candidate was underlined recently, when this serotype "22" was recognized in N.meningitidis B strain isolated from a died 10 months old boy. These results indicate the epidemiological and clinical significance of the new serotype candidate "22" in the Czech Republic.

[Cytotoxicity and invasive virulence of Yersinia enterocolitis strains in various mammalian cell lines]. / Cytotoxicita a invazivita virulentních kmenu Yersinia enterocolitica na ruzných savcích bunecných liniích.

The authors tested the suitability of 10 cell lines for assessment of the virulence of strains of Yersinia enterocolitica by the cytotoxicity test. Line LLC-MK2, HEp-2, HeLa, L-132 and L929 are most suitable. Lines LEP, 3T3, MDCK and McCoy are usable, line BHK-21 is unsuitable. On the same cell lines also the invasiveness of the virulent and non-virulent strains of Y. enterocolitica was assessed. For detection of the internalization of bacteria a modified method of double fluorescent staining was used which is simpler and more rapid. The invasiveness, similarly as the cytotoxicity of the virulent strain was proved on all tested cell lines, contrary to the non-virulent strain which was neither cytotoxic nor invasive. Using strains of Y. enterocolitica, isolated from faeces of patients with diarrhoea of infectious etiology or another bowel disease and from water, the cytotoxicity method was compared with recommended biochemical virulence tests used in this country: bond with Congo red, crystal violet, dependence on Ca2+ and pyrazinamidase activity. Strains of serotype 0:5, 0:6.31 and 0:7.8 biotype 1 gave in all tests negative results, strains of serotype 0:3 biotype 4 differed. The highest number of positive strains was detected by the cytotoxicity test. The positivity of strains of this serotype was assessed by the invasiveness test in vitro. The cytotoxicity test was thus proved to be a very effective, rapid and easy test of detection of the virulence of Y. enterocolitica strains.

Interaction of virulent and non-virulent Yersinia enterocolitica strains and an epithelial and a phagocytic cell lines.

Morphological alterations of HEp-2 and P338D1 cells were detected as result of Yersinia virulent strains action only. A non-virulent strain caused none of these alterations even 24 h post infection (p.i.). The internalization of the bacteria was demonstrated by double fluorescence staining. Adherence and beginning of cell invasion of virulent strains was detected 30 min p.i. already. Two hours p.i. these bacteria were in great numbers inside the cells of both lines. The non-virulent Yersinia strain was found only in the P338D1 macrophages at 2 and 24 h p.i. but in smaller numbers than virulent strains. Electron microscopy confirmed that internalization of virulent strains Y526 and Y527 was done by the phagocytosis of both cell lines. Even intracellular replication of these virulent strains was observed in both cell lines at 2 and 24 h p.i. Both bacterial strains were disintegrated as well as they multiplied inside the cells. In strain Y526 disintegration of bacteria prevailed, whereas their replication predominated in the strain Y527. At 24 h p.i. cells infected with strain Y527 were sac-like, with rudiments of the cytoplasm and organelles, packed with bacteria that were released after cell membrane rupture. Cells infected with strain Y526 were metabolically active and even at 24 h p.i. predominately contained disintegrated bacteria, but even in this case replication and release of bacteria was observed.

[A new look at transmission and circulation of tick encephalitis virus in nature]. / Nový pohled na prenos a cirkulaci viru klíst'ové encefalitidy v prírode.

The author summarizes briefly the results of experiments which provide evidence of non-viraemic transmission of the virus from infected to non-infected ticks during co-feeding on a non-viraemic host. This mode of transmission originally found in the Thogoto virus and its vector, the tick Rhipicephalus appendiculatus, was proved also in the virus of tick-borne encephalitis and its vector Ixodes ricinus. Based on this fact some conclusions are drawn revising the accepted view regarding the life cycle of tick-borne encephalitis virus in nature and in particular the relationship: vector-host-virus.