Leptospira interrogans Sensu Lato in Wild Small Mammals in Three Moravian Localities of the Czech Republic.

Leptospirosis is a widespread zoonosis, affecting humans, domestic animals and wildlife, with small mammals as a reservoir of this infection. In recent years, this disease has been re-emerging and affects approximately 1 million people all over the world each year. Due to this disease having a significant health impact, it is important to identify the source and method of infection. The risk of Leptospira sp. infection is higher mainly in the cities of developed and industrialised countries. The aim of the study was the detection of antibodies against Leptospira sp. in some wild small mammals captured in the Czech Republic. In total, samples of 855 animals captured in three locations of Moravia during a six-year study (2010-2015) were examined by a microscopic agglutination test, using eight serovars of Leptospira interrogans sensu lato, representing serogroups Grippotyphosa, Icterohaemorrhagiae, Australis, Canicola, Sejroe, Javanica, Pomona and Pyrogenes, as antigens. Antibodies to Leptospira sp. were detected in 6.1% (52/855) of animals, with a prevalence of 6.4% (51/801) and 1.9% (1/54) in rodents and insectivores, respectively. The only statistically significant difference (p ≤ 0.05) was in prevalence between individual species (0-33%), while there were no differences in sex (6.7% in females and 5.1% in males), locality (1.8-8%) and year of trapping (0-8.4%). Only two serovars, L. interrogans serovar Pomona and L. interrogans serovar Grippotyphosa, were detected in 5.5% and 0.5% of animals, respectively. The prevailing serovar of pathogenic L. interrogans s.l. can be identified in a number of infected people in the Czech Republic. The composition of vaccines should be based on the current occurrence of Leptospira serovars in the actual territory. For this reason, the occurrence of Leptospira and its serovars should therefore be regularly monitored.

Wild Small Mammals and Ticks in Zoos-Reservoir of Agents with Zoonotic Potential?

Wild small mammals and ticks play an important role in maintaining and spreading zoonoses in nature, as well as in captive animals. The aim of this study was to monitor selected agents with zoonotic potential in their reservoirs and vectors in a zoo, and to draw attention to the risk of possible contact with these pathogens. In total, 117 wild small mammals (rodents) and 166 ticks were collected in the area of Brno Zoo. Antibodies to the bacteria Coxiella burnetii, Francisella tularensis, and Borrelia burgdorferi s.l. were detected by a modified enzyme-linked immunosorbent assay in 19% (19/99), 4% (4/99), and 15% (15/99) of rodents, respectively. Antibodies to Leptospira spp. bacteria were detected by the microscopic agglutination test in 6% (4/63) of rodents. Coinfection (antibodies to more than two agents) were proved in 14.5% (15/97) of animals. The prevalence of C. burnetii statistically differed according to the years of trapping (p = 0.0241). The DNAs of B. burgdorferi s.l., Rickettsia sp., and Anaplasma phagocytophilum were detected by PCR in 16%, 6%, and 1% of ticks, respectively, without coinfection and without effect of life stage and sex of ticks on positivity. Sequencing showed homology with R. helvetica and A. phagocytophilum in four and one positive samples, respectively. The results of our study show that wild small mammals and ticks in a zoo could serve as reservoirs and vectors of infectious agents with zoonotic potential and thus present a risk of infection to zoo animals and also to keepers and visitors to a zoo.

Urinary shedding of leptospires in palearctic bats.

Leptospirosis is a bacterial zoonotic infection of worldwide occurrence. Bats, like other mammalian reservoirs, may be long-term carriers that maintain endemicity of infection and shed viable leptospires in urine. Direct and/or indirect contact with these Leptospira shedders is the main risk factor as regards public health concern. However, knowledge about bat leptospirosis in the Palearctic Region, and in Europe in particular, is poor. We collected urine from 176 specimens of 11 bat species in the Czech Republic, Poland, Republic of Armenia and the Altai Region of Russia between 2014 and 2019. We extracted DNA from the urine samples to detect Leptospira spp. shedders using PCR amplification of the 16S rRNA and LipL32 genes. Four bat species (Barbastella barbastellus n = 1, Myotis bechsteinii n = 1, Myotis myotis n = 24 and Myotis nattereri n = 1) tested positive for Leptospira spp., with detected amplicons showing 100% genetic identity with pathogenic Leptospira interrogans. The site- and species-specific prevalence range was 0%-24.1% and 0%-20%, respectively. All bats sampled in the Republic of Armenia and Russia were negative. Given the circulation of pathogenic leptospires in strictly protected Palearctic bat species and their populations, non-invasive and non-lethal sampling of urine for molecular Leptospira spp. detection is recommended as a suitable surveillance and monitoring strategy. Moreover, our results should raise awareness of this potential disease risk among health professionals, veterinarians, chiropterologists and wildlife rescue workers handling bats, as well as speleologists and persons cleaning premises following bat infestation.

Active surveillance for antibodies confirms circulation of lyssaviruses in Palearctic bats.

BACKGROUND: Palearctic bats host a diversity of lyssaviruses, though not the classical rabies virus (RABV). As surveillance for bat rabies over the Palearctic area covering Central and Eastern Europe and Siberian regions of Russia has been irregular, we lack data on geographic and seasonal patterns of the infection. RESULTS: To address this, we undertook serological testing, using non-lethally sampled blood, on 1027 bats of 25 species in Bulgaria, the Czech Republic, Poland, Russia and Slovenia between 2014 and 2018. The indirect enzyme-linked immunosorbent assay (ELISA) detected rabies virus anti-glycoprotein antibodies in 33 bats, giving an overall seroprevalence of 3.2%. Bat species exceeding the seroconversion threshold included Myotis blythii, Myotis gracilis, Myotis petax, Myotis myotis, Murina hilgendorfi, Rhinolophus ferrumequinum and Vespertilio murinus. While Myotis species (84.8%) and adult females (48.5%) dominated in seropositive bats, juveniles of both sexes showed no difference in seroprevalence. Higher numbers tested positive when sampled during the active season (10.5%), as compared with the hibernation period (0.9%). Bat rabies seroprevalence was significantly higher in natural habitats (4.0%) compared with synanthropic roosts (1.2%). Importantly, in 2018, we recorded 73.1% seroprevalence in a cave containing a M. blythii maternity colony in the Altai Krai of Russia. CONCLUSIONS: Identification of such "hotspots" of non-RABV lyssavirus circulation not only provides important information for public health protection, it can also guide research activities aimed at more in-depth bat rabies studies.

Seroprevalence of Antibodies against Borrelia burgdorferi s. l. and Leptospira interrogans s. l. in Cats in district of Brno and its environs, the Czech Republic.

OBJECTIVES: The aim of this study is to evaluate the seroprevalence of antibodies of Borrelia burgdorferi sensu lato (Bbsl) and Leptospira interrogans sensu lato (Lisl) and their possible concurrence in domestic cats living in variable conditions in South Moravia in the district of Brno and its environs. Additional objectives were to discover possible differences in seroprevalence between groups of cats living in different living conditions, and to determine the spectrum of Leptospira serogroups in cats in the same places. MATERIAL AND METHODS: A total of 360 blood sera from domestic cats of 3 different sets were collected during the period 2013-2015. All samples were examined using ELISA for the detection of IgM and IgG antibodies against Bbsl, and the microscopic agglutination test (MAT) for the detection of antibodies against 8 serogroups of Lisl. RESULTS: The ELISA method determined 15.8%, 4.8% and 10.3% IgM anti-Borrelia antibodies in the patient group, shelter cats and street cats, respectively. IgG anti-Borrelia antibodies were found in 6.2%, 9.5%, 5.2%, respectively. Antibodies specific for 5 Leptospira serogroups were detected by the use of MAT in 8.8%, 9.5% and 10.3% of cats from the investigated groups. The total positivity of all examined cats for anti-Borrelia antibodies was 18.0% and for anti-Leptospira - 9.2%. CONCLUSIONS: Cats can be infected with both Bbsl and Lisl. The obtained results are exclusive to the city of Brno and its environs, and are comparable to the limited previous studies. There is a need for further studies of clinical signs of both infections and the possible transmission of Leptospira by ticks.

West Nile Virus equine serosurvey in the Czech and Slovak republics.

A serological survey for West Nile virus (WNV) infection involved 395 horses from 43 administrative districts of the Czech Republic (163 animals) and 29 districts of Slovakia (232 animals), sampled between 2008 and 2011. Using a plaque-reduction neutralization microtest, antibodies to WNV were not detected in any horse from the Czech Republic, whereas 19 nonvaccinated horses from Slovakia had specific antibodies to WNV (no cross-reactions were observed with tick-borne encephalitis and Usutu flaviviruses in those animals). The seropositivity rate of nonvaccinated horses in Slovakia was 8.3% (95% confidence interval [CI] 4.7-11.9%), and autochthonous local infection with WNV occurred at least in 11, i.e., 4.8% (95% CI 2.0-7.6%) of the animals. All seropositive horses lived in six lowland districts of southern Slovakia; overall, 15.1% (95% CI 8.8-21.4%) of 126 nonvaccinated horses were seropositive in those districts, situated relatively closely to the border with Hungary, i.e., the country where WNV disease cases have been reported in birds, horses and humans since 2003.

Lead toxicosis of captive vultures: case description and responses to chelation therapy.

BACKGROUND: Lead, a serious threat for raptors, can hamper the success of their conservation. This study reports on experience with accidental lead intoxication and responses to chelation therapy in captive Cinereous (Aegypius monachus) and Egyptian (Neophron percnopterus) Vultures. RESULTS: Soil contamination by lead-based paint sanded off the steel aviary resulted in poisoning of eight Cinereous and two Egyptian Vultures. A male Egyptian Vulture developed signs of apathy, polydipsia, polyuria, regurgitation, and stupor, and died on the next day. Liver, kidney and blood lead concentrations were 12.2, 8.16 and 2.66 µg/g, respectively. Laboratory analyses confirmed severe liver and kidney damage and anaemia. Blood Pb levels of Pb-exposed Cinereous Vultures were 1.571 ± 0.510 µg/g shortly after intoxication, decreased to 0.530 ± 0.165 µg/g without any therapy in a month and to 0.254 ± 0.097 µg/g one month after CaNa(2)EDTA administration. Eight months later, blood lead levels decreased to close to the background of the control group. Blood parameters of healthy Pb-non-exposed Cinereous Vultures were compared with those of the exposed group prior to and after chelation therapy. Iron levels in the lead-exposed pre-treatment birds significantly decreased after chelation. Haematocrit levels in Pb-exposed birds were significantly lower than those of the controls and improved one month after chelation. Creatine kinase was higher in pre-treatment birds than in the controls but normalised after therapy. Alkaline phosphatase increased after chelation. A marked increase in the level of lipid peroxidation measured as thiobarbituric acid reactive species was demonstrated in birds both prior to and after chelation. The ferric reducing antioxidant power was significantly lower in pre-treatment vultures and returned to normal following chelation therapy. Blood metallothionein levels in lead-exposed birds were higher than in controls. Reduced glutathione dropped after CaNa(2)EDTA therapy, while oxidised glutathione was significantly lower in both pre- and post-treatment birds. A chick in an egg produced by a Cinereous Vulture female two months after lead toxicosis died on day 40 of artificial incubation. Lead concentrations in foetal tissues were consistent with levels causing avian mortality. CONCLUSIONS: The reported blood parameters and reproduction impairment in captive birds may have implications for professionals dealing with lead exposure in wild birds.

Risk of combined exposure of birds to cyanobacterial biomass containing microcystins, acetylcholinesterase inhibitor and anticoagulant.

OBJECTIVES: The objective of this study was to examine the hypothesis that a combination of cyanobacterial biomass containing microcystins, acetylcholinesterase inhibitor and anticoagulant can enhance avian toxic effects produced by single exposures only. METHODS: A total of 48 two-month-old Japanese quails (Coturnix coturnix japonica) with average body weight of 160 g were randomly divided into 8 experimental groups of six birds and sex ratio of 1:1. Experimental groups of control Japanese quails (C) and birds exposed to single and combined sub-lethal doses of paraoxon (P), bromadiolone (B), and microcystins in cyanobacterial biomass (M) included: C, P, P+B, B, B+M, P+M, M, and P+B+M. During the 10-day exposure birds in the respective groups received biomass containing 61.62 µg microcystins daily (i.e. 26.54 µg MC-RR, 7.62 µg MC-YR and 27.39 µg MC-LR), two 250 µg/kg doses of paraoxon, and two 500 mg/kg doses of bromadiolone. Group responses were compared using standard plasma biochemistry and antioxidant/oxidative stress parameters in tissues. RESULTS: While single and double combinations of toxicants induced responses in individual biochemical parameters measured and evaluated using univariate statistical analysis, those in the triple exposure were most extensive. The principal component analysis of antioxidant/oxidative stress parameters (glutathione reductase, lipid peroxidation, and ferric reducing antioxidant power) in tissues (liver, kidney, heart, brain, lungs, gonads, and pectoralis major muscle) clearly separated the triple group (P+B+M) from all single and double exposure groups and the control and indicated thus marked joint effects in the overall pattern of antioxidant/oxidative stress responses of this group. The separation was driven by the modification of the ferric reducing antioxidant power levels in heart and brain and the cardiac lipid peroxidation level, in particular. CONCLUSIONS: This experiment contributes to the understanding of the pathogenic mechanisms of combined sub-lethal exposure to natural toxins and agrochemicals and may be used for risk assessment of environmental pollution in birds.

Avian high-dose toxicity of cyanobacterial biomass.

OBJECTIVES: Previous studies using oral administration of environmentally relevant doses of cyanobacterial biomass containing microcystins (MCs) induced only sub-lethal effects in experimental birds. Therefore, the objective of this study was to obtain data on avian high-dose toxicity of MCs and compute LD50, if possible, following the natural oral route of administration. DESIGN: Responses of birds to single high-dose exposure to MCs were evaluated in fourteen-day old Japanese quail males (Coturnix coturnix japonica) with average body weight of 50 g which were randomly divided into five groups. Birds from four experimental groups were administered 7.5 ml of cyanobacterial biomass suspension containing increasing MCs quantities of 2500, 5000, 10000, and 20000 µg/kg using oral gavage. Controls received an equal dose of drinking water instead of the test substance. Birds were observed for clinical signs of acute toxicity. Survivors were killed on day 5 to obtain body and liver weights. A five-grade semi-quantitative system for histopathological liver damage scoring was used to compare cyanobacterial-biomass-exposed birds against controls. RESULTS: No mortality occurred during the period of five days post exposure in both control and MCs-exposed groups and this high-dose experiment failed to provide data to compute the LD50. Nevertheless, marked sub-lethal effects were recognised in the damage of liver that included dose-dependent changes in the body/liver ratios and morphological changes ranging from mild vacuolar dystrophy to focal liver necroses in the highest exposure group. Hepatic lesions were mainly observed in the pericentral area of the liver. CONCLUSIONS: Though maximum cyanobacterial biomass dose rates that could be administered to birds of the size were used in the present experiment and more pronounced hepatic lesions than after exposure to environmentally relevant doses were observed, birds would probably have survived unless killed for histopathology on day 5 of exposure. These results provide support to previously reported data on sub-lethal effects following exposure to cyanobacterial biomass containing MCs in birds and mortality occurring only in birds under combined action with other stressors.

Effects of sublethal exposure of European brown hares to paraoxon on the course of tularemia.

OBJECTIVES: The causative agent of tularemia Francisella tularensis is highly infectious and lagomorphs are important reservoirs and a source of human disease. The aim of the present study was to test the hypothesis that sublethal exposure to pesticides increases the susceptibility of hares to F. tularensis and modulates the course of the infection. METHODS: Experimental hares were allocated to a) control, b) paraoxon-treated, c) F. tularensis-treated, and d) paraoxon-and-F. tularensis-treated groups of five specimens on a random basis and subcutaneously inoculated with a wild F. tularensis subsp. holarctica strain (a single dose of 9 × 108 CFU pro toto) and/or injected a sublethal dose of paraoxon (100 µg/kg). Group differences were evaluated using survival curves, oxidative stress responses as well as caspase-3 and acetylcholinesterase activities in whole blood samples collected on day 2 post exposure. RESULTS: The paraoxon-and-F. tularensis-treated group showed a rapid onset of clinical signs and all deaths occurred on days 2 and 3 post exposure. F. tularensis-inoculated hares survived from 3 to 10 days, while only one hare died on day 12 in the paraoxon-treated group. Survival curves in the three exposed groups were significantly different from the control and median survival in F. tularensis-inoculated and paraoxon-and-F. tularensis-treated hares amounted to 7 and 2 days, respectively. Compared with controls, significant responses included an eight- and seven-fold activation of caspase-3 in F. tularensis-inoculated and paraoxon-and-F. tularensis-treated hares, respectively, and a 1.5-fold decrease of blood acetylcholinesterase activities in the paraoxon-treated and paraoxon-and-F. tularensis-treated groups. There was a 1.3- to 1.4-fold decrease of the ferric reducing antioxidant power in blood of F. tularensis-inoculated hares and the paraoxon-and-F. tularensis-treated group, respectively. The blood lipid peroxidation levels were of no differences among the four experimental groups. CONCLUSIONS: Results of this study can help understand the pathogenesis of tularemia and mortality of hares in agricultural habitats. Use of anticholinesterase agents in agriculture can pose a threat of infectious disease outbreaks and higher mortality in wildlife populations.

Mycoplasma gallisepticum infection in the grey partridge Perdix perdix: outbreak description, histopathology, biochemistry and antioxidant parameters.

BACKGROUND: The grey partridge is an important game bird in Europe that has declined considerably over the last decades. The production and release of farm-bred birds can be threatened by infectious agents. The objective of this study was to describe the outbreak, pathology, and blood and tissue biochemical responses in a flock of grey partridges naturally infected with Mycoplasma gallisepticum. RESULTS: Morbidity and mortality rates were 100% and 60%, respectively. Necropsy revealed an accumulation of caseous exudate within the infraorbital sinuses, tracheitis, pneumonia and airsacculitis. There were significant increases in activities of lactate dehydrogenase, creatine kinase and amylase, and levels of total protein and glucose in Mycoplasma-infected birds when compared to control. Catalase showed significantly lower activity in the heart, lungs, liver and gonads of Mycoplasma-infected birds. Glutathione-S-transferase activity was elevated in the eye and the associated infraorbital sinus and kidneys, and decreased in the liver. Decreased levels of reduced glutathione were found in the heart, kidneys, liver and gonads. The activity of glutathione reductase was lower only in the lungs. Compared to healthy birds, mycoplasmosis in the grey partridge caused significant differences in the level of lipid peroxidation in lungs and plasma (p < 0.05), while the ferric reducing antioxidant power was lower in the heart and kidneys (p < 0.01). Significant correlations among responses of the antioxidant parameters were found namely in the heart, lungs, spleen, liver and plasma. There were also numerous significant inter-tissue correlations of all the studied antioxidant parameters. CONCLUSIONS: The present study demonstrates the high susceptibility of grey partridges to natural infection by M. gallisepticum, the severity of the disease based on histopathology, and the modulation of blood chemical profiles and oxidative stress-associated parameters in the avian hosts, thus enhancing the understanding of the pathogenesis of mycoplasmosis in birds. Moreover, the reported reference values can be useful for the evaluation of the state of health in grey partridges.

Biochemical responses and oxidative stress in Francisella tularensis infection: a European brown hare model.

BACKGROUND: The aim of the present study was to investigate biochemical and oxidative stress responses to experimental F. tularensis infection in European brown hares, an important source of human tularemia infections. METHODS: For these purposes we compared the development of an array of biochemical parameters measured in blood plasma using standard procedures of dry chemistry as well as electrochemical devices following a subcutaneous infection with a wild Francisella tularensis subsp. holarctica strain (a single dose of 2.6 × 109 CFU pro toto). RESULTS: Subcutaneous inoculation of a single dose with 2.6 × 109 colony forming units of a wild F. tularensis strain pro toto resulted in the death of two out of five hares. Plasma chemistry profiles were examined on days 2 to 35 post-infection. When compared to controls, the total protein, urea, lactate dehydrogenase, aspartate aminotransferase and alanine aminotransferase were increased, while albumin, glucose and amylase were decreased. Both uric and ascorbic acids and glutathione dropped on day 2 and then increased significantly on days 6 to 12 and 6 to 14 post-inoculation, respectively. There was a two-fold increase in lipid peroxidation on days 4 to 8 post-inoculation. CONCLUSIONS: Contrary to all expectations, the present study demonstrates that the European brown hare shows relatively low susceptibility to tularemia. Therefore, the circumstances of tularemia in hares under natural conditions should be further studied.

Neospora caninum and Toxoplasma gondii antibodies in European brown hares in the Czech Republic, Slovakia and Austria.

European brown hares (Lepus europaeus) coming from the Czech Republic, Slovakia and Austria were tested for serum antibodies against Neospora caninum by a competitive-inhibition enzyme-linked immunosorbent assay and for serum antibodies against Toxoplasma gondii by an indirect fluorescent antibody test. In a total of 925 samples, 280 (30%) and 132 (14%) reacted positively for N. caninum and T. gondii antibodies, respectively. Out of 333 hares in the Czech Republic, 129 (39%) and 71 (21%) reacted positively for N. caninum and T. gondii antibodies, respectively. Out of 209 hares in Slovakia, 8 (4%) and 13 (6%) reacted positively for N. caninum and T. gondii antibodies, respectively. Out of 383 hares in Austria, 143 (37%) and 48 (13%) reacted positively for N. caninum and T. gondii antibodies, respectively. Mixed infection (concurrent presence of both N. caninum and T. gondii antibodies) was found in 25 (8%) hares in the Czech Republic, in 14 (4%) hares in Austria and in none hare in Slovakia. It is the first report of antibodies against Neospora caninum in hares in the Czech Republic and Austria.

Assessment of low-molecular-weight antioxidants in Francisella tularensis infected hosts: comparison of two rodents with different susceptibility to tularemia.

OBJECTIVES: Bacterium Francisella tularensis is the causative agent of tularemia disease. It is a zoonosis accompanied with high mortality when untreated. Small rodents and hares, in particular, are natural reservoirs of tularemia. Despite physiological similarity of common hosts, tularemia exerts different mortality rates. The pathogenesis of tularemia is still not fully understood. The main pathway is associated with proliferation in macrophages after activation by reactive oxygen species in phagosomes. DESIGN: A fully virulent strain of F. tularensis subsb. holarctica was used for infection of laboratory BALB/c mice (Mus musculus) and common voles (Microtus arvalis) representing murine and microtine species. The total level of low-molecular- weight antioxidants (LMWA) in plasma was assayed by cyclic voltammetry. RESULTS: It was found that common voles are more resistant to tularemia progression when compared to mice. When LMWA assayed, surprising changes in LMWA levels were found. Both mice and common voles were infected with high dose resulting in overall mortality. While there was a quick depletion of LMWA in plasma in mice, common voles were even able to increase LMWA. CONCLUSION: It seems that LMWA play an important role in the organism s protection during tularemia. The ability to compensate the LMWA losses and increase levels of antioxidants in common voles is probably responsible for its lower susceptibility to tularemia.

Tularemia induces different biochemical responses in BALB/c mice and common voles.

BACKGROUND: Both BALB/c mice and common voles (Microtus arvalis) are considered highly susceptible to tularemia. However, the common vole is reported to harbour Francisella tularensis in European habitats as well as to survive longer with chronic shedding of the bacterium. The purpose of the present study was to compare the response of these two rodents to a wild Francisella tularensis subsp. holarctica strain infection. METHODS: Rodents were evaluated for differences in the total antioxidant capacity derived from low-molecular-weight antioxidants, biochemistry including lipid metabolism, tissue bacterial burdens and histopathology following experimental intraperitoneal infection with 160 colony forming units (CFU) pro toto. RESULTS: Bacterial burdens in common voles started to develop later post-exposure and amounted to lower levels than in BALB/c mice. Elevation of liver function enzymes was more pronounced in mice than common voles and there were marked differences in lipid metabolism in the course of tularemia in these two species. Hypertriglyceridemia and hypercholesterolemia developed in mice, while physiologically higher levels of triglycerides and cholesterol showed a decreasing tendency in common voles. On the other hand, the total plasma antioxidant capacity gradually dropped to 81.5% in mice on day 5 post-infection, while it increased to 130% on day 6 post-infection in common voles. Significant correlations between tissue bacterial burdens and several biochemical parameters were found. CONCLUSION: As differences in lipid metabolism and the total antioxidant capacity of highly susceptible rodent species were demonstrated, the role of triglycerides, cholesterol and antioxidants in tularemic sepsis should be further investigated.

Piezoelectric Biosensor for a Simple Serological Diagnosis of Tularemia in Infected European Brown Hares (Lepus europaeus).

Piezoelectric biosensor was used for diagnosis of infection by Francisellatularensis subsp. holarctica in European brown hares. Two kinds of experiments wereperformed in this study. First, sera from experimentally infected European brown hares(Lepus europaeus) were assayed by piezoelectric biosensor and the seventh day postinfection was found as the first one when statistically significant diagnosis of tularemia waspossible; all other sera collected from hares later than on day 7 following the infection werefound tularemia positive. Typing to classify the field strain of F. tularensis used for theexperimental infection was confirmed by proteome study. Second, sera from 35 Europeanbrown hare specimens sampled at hunting grounds and tested as tularemia positive by slowagglutination allowed diagnosis of tularemia by the piezoelectric biosensor. All these sera ofnaturally infected hares were found as tularemia positive, too. Efficacy of the piezoelectricbiosensor for the serological diagnosis of tularemia is discussed.

Ecological conditions of natural foci of tularaemia in the Czech Republic.

Tularaemia, a zoonosis of veterinary and public health importance, commonly occurs in the Czech Republic as well as other countries of Northern Hemisphere. The objective of this study was to analyse the environmental conditions of distribution of natural foci of tularaemia and their long-term persistence in the Czech Republic. A geographic information system has been used for this purpose. A new variable (chi(t)), the mean number of natural foci in a specific area, has been suggested for the evaluation of diseases occurring in natural foci. Comparing two 15-year periods, a close correlation between the geographic distribution and numbers of natural foci of tularaemia in the Czech Republic in 1971-1985 and 1986-2000 (r = 0.91, n = 1814, t = 92.50, p = 0.01) was found. Natural foci of tularaemia have been persistent, but not stationary, over the period of 30 years and the geographic area of their occurrence has not been considerably growing or diminishing in the Czech Republic. The highest numbers of natural foci of tularaemia were in habitats of alluvial forests (chi(t) = 7.20), geographic areas of up to 200 m of elevation above sea (chi(t) = 9.18), 8.1-10.0 degrees C of mean annual air temperature (chi(t) = 6.24), 450-700 mm of mean annual precipitation (chi(t) = 2.84), and 2001-2200 hour of mean annual sunshine duration (chi(t) = 8.77). It was proved that tularaemia persists in specific areas of natural foci, the general environmental conditions of which can be defined and make it possible to predict the occurrence of tularaemia in other areas of suitable conditions.