[The incidence of tularemia in Slovakia in 1997-2008]. / Výskyt tularémie na Slovensku v rokoch 1997-2008.

STUDY OBJECTIVE: Analysis of epidemiological trends in tularemia in the Slovak Republic (SR) over the last 12 years and changes in the epidemiology of this infection. MATERIAL AND METHODS: The sources of data for the epidemiological analysis are the SR annual tularemia surveillance report, records of epidemiological investigation of reported cases and knowledge from long-term surveillance of natural foci. The incidence of tularemia was analyzed based on the standard epidemiological indicators and in relation to infection sources and transmission routes. RESULTS: In 1997-2008, a total of 453 tularemia cases were reported from all administrative regions in Slovakia, the mean incidence was 0.71/10(5) population and 95.4% of cases were reported in the West, with the predominance of cases in the Nitra region where the mean incidence was 4/10(5) population. In the outbreak of 2002, the incidence of tularemia was as high as 18 cases/10(5) population. Tularemia cases were reported in all age groups and more than half of the patients were aged between 30 to 59 years. Tularemia cases were 1.9 times more frequent in males than in females. The seasonal trend peaked in summer, month of July. In a greater proportion of cases, 58.5%, the source of infection was other than brown hares, 16.3%, which correlates well with the decrease in the hare tularemia foci in the study period. The most frequent route of transmission was work exposure to contaminated animal feed and bedding and in an dusty environment with increased occurrence of small rodents. The proportion of tick-borne and biting insect-borne cases was 12.8% and 12.4% of cases were not elucidated epidemiologically. Clinical manifestations were ulceroglandular and glandular tularemia in 55.6%, pulmonary tularemia in 21.2 %, oroglandular tularaemia in 18.8% and other forms of tularaemia in 4.4%. CONCLUSION: The changes in the distribution of sources of infection and routes of transmission resulted in changed epidemiological characteristics and clinical forms of tularemia in Slovakia. The importance of tularemia surveillance is underlined.

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.

Evaluation of PCR-based methods for discrimination of Francisella species and subspecies and development of a specific PCR that distinguishes the two major subspecies of Francisella tularensis.

Previous studies have demonstrated that the four subspecies of the human pathogen Francisella tularensis, despite showing marked variations in their virulence for mammals and originating from different regions in the Northern Hemisphere, display a very close phylogenetic relationship. This property has hampered the development of generally applicable typing methods. To overcome this problem, we evaluated the use of PCR for discrimination of the subspecies using various forms of long arbitrary primers or primers specific for repetitive extragenic palindromic sequences (REP) or enterobacterial repetitive intragenic consensus (ERIC) sequences. Patterns generated by use of REP, ERIC, or long arbitrary primers allowed differentiation at the species level and of the four subspecies of F. tularensis. With each of these three methods, similar or identical clustering of strains was found, and groups of strains of different geographical origins or differing in virulence showed distinct patterns. The discriminatory indices of the methods varied from 0.57 to 0.65; thus, the patterns were not sufficiently discriminatory to distinguish individual strains. The sequence of a fragment generated by amplification with an arbitrary primer was determined, and a region showing interstrain heterogeneity was identified. Specific primers were designed, and a PCR was developed that distinguished strains of F. tularensis subsp. holarctica from strains of other F. tularensis subspecies, including strains of the highly virulent F. tularensis subsp. tularensis. Notably, one European isolate showed the genetic pattern typical of the highly virulent F. tularensis subsp. tularensis, generally believed to exist only in North America. It is proposed that a combination of the specific PCR together with one method generating subspecies-specific patterns is suitable as a rapid and relatively simple strategy for discrimination of Francisella species and subspecies.

[A tularemia epidemic in western Slovakia 1995-1996]. / Epidémia tularémie na západnom Slovensku v rokoch 1995-1996.

The activation of natural foci of tularaemia in West Slovakia during 1994-1996 led to an epidemic outbreak in 1995-1996--mean annual morbidity 6.2 per 10(5) population. In comparison with the mean annual morbidity rate in the preceding period (1980-1994), a more than sevenfold increase was recorded. Of 213 notified cases of the disease 156 cases occurred in 1995--morbidity 8.9 per 10(5) population--being the highest morbidity recorded in this endemic region since the period of epidemic occurrence in the 60s. The highest proportion of cases (59.2%) was recorded in the districts of Nitra and Nové Zámky. The activation of natural foci along with changing social conditions, caused also marked changes of some epidemiological characteristics of tularaemia in Slovakia, such as seasonal and professional occurrence, as well as clinical forms of the disease. The impaired epidemiological situation in the occurrence of tularaemia is pointing to the importance of systematic surveillance to improve the diagnosis of the disease and ensure effective preventive measures.

First isolation of Francisella tularensis subsp. tularensis in Europe.

Francisella tularensis subsp. tularensis is the common causal agent of tularemia in the USA and Canada, while F. tularensis subsp. palaearctica (holarctica) occurs in Europe, Asia, and to a minor extent in North America. F. tularensis subsp. mediaasiatica was found only in central Asia in a part of the former Soviet Union. Of the total of 155 F. tularensis strains isolated over the years 1978-1996 during the surveillance of tularemia in Slovakia, 65 were from small mammals, 68 from ticks and 22 from mites and fleas. They were characterized and classified by basic markers of infraspecific taxonomy in tests in vitro and compared with type strains of three subspecies and biovars of F. tularensis. Comparative studies have revealed biological properties characteristic of F. tularensis subsp. tularensis in 17 strains isolated from fleas and mites parasiting on small terrestrial mammals, collected in the Danube region, near Bratislava. These strains fermented glycerol, glucose, were positive for citrulline ureidase and sensitive to erythromycin, in contrast to the other 138 isolates classified as F. tularensis subsp. palaearctica (holarctica), biovar II, which fermented only glucose, were negative for citrulline ureidase and resistant to erythromycin. Two selected pairs of isolates with properties characteristic of F. tularensis subsp. palaearctica (holarctica), biovar II (SE-210, SE-234) and of F. tularensis subsp. tularensis (SE-219, SE-221), as shown in tests in vitro, were further examined for their pathogenicity on white mice, guinea pigs and domestic rabbits. In tests of virulence on domestic rabbits, the isolates SE-210 and SE-234 had low pathogenicity, while the isolates SE-219 and SE-221 exhibited high pathogenicity, which along with their biochemical properties confirmed their identification as strains of F. tularensis subsp. tularensis. The first findings of the highly virulent strains of F. tularensis subsp. tularensis in Europe indicate a serious event from epidemiologic and epiozootologic aspects, requiring systematic surveillance.

[Analysis of the incidence and routes of transmission of tularemia in Slovakia]. / Analýza výskytu a transmisívneho prenosu tularémie na Slovensku.

The morbidity from tularaemia in Slovakia has since the epidemic incidence in the sixties a declining trend. The mean annual morbidity rate declined from 5.5 per 10(5) population in 1960-1969 to 0.3 in 1980-1994 and is markedly influenced by the incidence of tularaemia in the well known epidemic area in western Slovakia where in 1985 also the first cases of transmission of tularaemia by ticks were recorded in Slovakia. During the last decade (1985-1994) in Slovakia 126 cases were notified, out of them 96.8% in the western Slovakian region, more than half of the cases were recorded in the districts of Nitra and Nové Zámky. The analysis of the incidence of the disease in western Slovakia as compared with the previous decade (1975-1984) drew attention to marked changes in the epidemiology of tularaemia and a rise of the cases transmitted from other sources of infection than hares and by ectoparasites. Their ratio is almost 66%, from that 20.5% formed by cases transmitted by ticks (15.6%) and by other arthropods (4.9%). In the majority of patients with ticks in the case-history the ulceroglandular form of the disease was recorded (79%), with the primary affection at the site of the tick-bite, in the majority with lymphadenitis in the inguinal area (63.2%). The majority of the cases was recorded in summer, most frequently in occupational groups: pupils and students, forestry workers and mainly other occupations. The results of an epidemiological survey and data on the prevalence of for ticks infected with Francisella tularensis in the endemic area of Slovakia indicate that the tick-borne of tularaemia in humans Central European ecological conditions is probably more frequent than hitherto assumed.

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.

Mixed natural focus of tick-borne encephalitis, tularemia and haemorrhagic fever with renal syndrome in west Slovakia.

Total of 923 small mammals of 7 species were collected in locality Záhorská Ves, West Slovakia, in 1990-1992. Among examined small mammal species it was Clethrionomys glareolus (48.7% of total, 17.5% positive for tick-borne encephalitis (TBE) virus antibodies), Apodemus flavicollis (29.7% of total, 17.5% positive), A. sylvaticus (11.3% of total, 16.3% positive), and Microtus arvalis (6.2% of total, 10.5% positive). The most abundant tick species (larval and nymphal stages) on small mammals was Ixodes ricinus. The extensity of infestation was 35.1-50.7%, and the intensity of infestation ranged in average from 4.1 to 7.8 ticks per animal. Out of 884 small rodent serum samples 16.9% had neutralizing antibody to TBE virus. Eight TBE virus isolates were recovered, six from C. glareolus and one each from A. flavicollis and A. sylvaticus; seven isolates were from brain tissue and one was from a pool of lung and liver tissues. One strain of Francisella tularensis was isolated from a pool of spleens of four C. glareolus collected in August 1991. Hantavirus antigens were detected in lung tissues of four M. arvalis collected in July and November 1990-1992. Antibody to Hantaan virus was detected by ELISA in one serum sample of A. flavicollis (titer 1:256) and antibody to Puumala virus in one serum sample of C. glareolus (titer 1:16).

[Surveillance of Francisella tularensis infection in dogs in Bratislava]. / Sledovanie premorenia bratislavských psov Francisella tularensis.

Out of 548 serologically investigated dogs from Bratislava and other regions of Slovakia and Moravia, antibodies to F. tularensis were found in 16.4% (Tabs. I, II). In all the investigated groups of dogs from the region of Bratislava the highest seroprevalence by F. tularensis was recorded in watch dogs kept on farms and in cooperatives--37.5% and in rambling dogs--20.7% (Tab. I). The highest seropositivity was found in one to three year old dogs--22.2% (Tab. III). A similar degree of seroprevalence was also observed in one to three years old police dogs which came from the endemic region of tularemia--West Slovakia (19.3%) and East Slovakia (25.6%)--Tab. IV. These facts indicate the persistence of active natural foci in these regions. Serological investigations of the relatively great number of dogs from different regions of Slovakia showed that the presence of F. tularensis antibodies in this animal species, mainly in the watch dogs group, can be taken as a convenient marker or indicator of the existence of active natural foci of tularemia and as a suitable component for surveillance of this diseases.

[Infestation of ticks with Borrelia burgdorferi and Francisella tularensis in Slovakia]. / Stúdium premorenosti kliest'ov Boréliou burgdorferi a Francisellou tularensis na Slovensku.

Examination of ticks for the presence of F. tularensis resulted in the isolation of 29 strains, predominantly from the species D. reticulatus (19x), further from I. ricinus (8x) and from H. concinna (2x). The results confirmed the activity of the known endemic focus in the area of Podunajské Biskupice and revealed three new foci in the localities Smolenice (district of Trnava), Olichov (district of Nitra), and Plást'ovce (district of Levice). In the focus of Podunajské Biskupice the mean infestation of ticks of the species D. reticulatus was around 1.4% over the years of investigation. With regard to the one-year developmental cycle, D. reticulatus can be considered an appropriate indicator of current epizootic activity of the focus, while the occurrence of I. ricinus is rather an indicator of the geographic spread of foci. The third part of the study addresses questions of the ecology of ticks. It analyzes their biological characteristics in our conditions, their spread and bionomy, particularly from aspects related with their epidemiological significance as vectors of Lyma borreliosis and tularemia. Systematic follow up of infestation of ticks with causative agents of borreliosis and tularemia in established endemic foci as well as in epidemiologically indicated localities is an inevitable part of modern surveillance of these infections.

[Effort to eradicate a focus of tularemia and leptospirosis]. / Versuch der Tilgung eines Naturherdes der Tularämie und der Leptospirose.

An endemic area of tularemia and field fever was submitted to a complex surveillance over a period of more than 10 years. In 1977 an epizootic outbreak of tularemia appeared in all four localities studied. The infection rate in small mammals exceeded 10%. F. tularensis was isolated also from different species of ectoparasites. During 1978 the epizootic stopped in 3 foci, but continued to spread in one. Rodenticides (zinc phosphide) were therefore applied in this focus twice annually for 2 1/2 years. Since 1978, tularemia infections disappeared in small mammals as well as in man. The application of rodenticides did not influence the circulation of Leptospira grippotyphosa in small mammals. Their infection rate ranged between 3 and 6%. The reason for this striking difference is seen in the different pathogenesis and transmission of both infections.