Coxiella burnetii and Francisella tularensis in wild small mammals from the Czech Republic

Journal Pre-proof Coxiella burnetii and Francisella tularensis in wild small mammals from the Czech Republic ´ ´ Hana Lya Kuˇcerov, Alena Zˇ akovsk ´ ´ Marie Bud´ıkova, ´ Eva Bartov a, a, Helena Nejezchlebova´

PII:

S1877-959X(19)30072-X

DOI:

https://doi.org/10.1016/j.ttbdis.2019.101350

Reference:

TTBDIS 101350

To appear in:

Ticks and Tick-borne Diseases

Received Date:

19 February 2019

Revised Date:

8 November 2019

Accepted Date:

28 November 2019

´ ´ Please cite this article as: Bartov a´ E, Kuˇcerov HL, Zˇ akovsk a´ A, Bud´ıkova´ M, Nejezchlebova´ H, Coxiella burnetii and Francisella tularensis in wild small mammals from the Czech Republic, Ticks and Tick-borne Diseases (2019), doi: https://doi.org/10.1016/j.ttbdis.2019.101350

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Coxiella burnetii and Francisella tularensis in wild small mammals from the Czech Republic

Eva Bártová1*, Hana Lya Kučerová,2 Alena Žákovská,2,3, Marie Budíková4, Helena Nejezchlebová2

University of Veterinary and Pharmaceutical Sciences, Faculty of Veterinary Hygiene and

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1

Ecology, Department of Biology and Wildlife Diseases, Palackého tř. 1946/1, Brno, 612 42, Czech Republic

Masaryk University, Faculty of Science, Department of Comparative Animal Physiology and

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2

General Zoology, Kamenice 753/5, Brno, 625 00, Czech Republic

Masaryk University, Faculty of Education, Department of Biology, Kamenice 753/5, Brno,

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625 00, Czech Republic

Masaryk University, Faculty of Science, Department of Mathematics and Statistics,

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Kotlářská 2, Brno, 611 37, Czech Republic

*

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Corresponding author: Phone: +420 541562633; E-mail: [email protected] (E. Bártová)

ABSTRACT:

Wild rodents are an important source of the tick-borne pathogens Coxiella burnetii and

Francisella tularensis. The aim of our study was to assess the prevalence of antibodies and possible coexistence of these pathogens in wild small mammals from three localities in the Czech Republic. A total of 614 wild small mammals (324 Apodemus flavicollis, 145 Myodes

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glareolus, 50 Sorex araneus, 48 A. sylvaticus, 40 A. agrarius, six Microtus arvalis and one Talpa europaea) were trapped between 2012 and 2015. Their sera or heart extracts were examined by modified indirect enzyme-linked immunosorbent assay, with the detection of antibodies against C. burnetii and F. tularensis in 12% and 7% of animals, respectively; coinfection was identified in 4.4% of animals. The prevalence of C. burnetii and F. tularensis antibodies statistically differed according to animal species and sex (p < 0.05); the seroprevalence of C. burnetii (p < 0.05) also differed in the sampling period. The highest

glareolus (24% and 14%, respectively).

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Keywords: Apodemus; Myodes; Q fever; serology; tularemia

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prevalence of antibodies against C. burnetii and F. tularensis was detected in the case of M.

Introduction

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Q fever is a widespread zoonotic disease caused by Coxiella burnetii, a ubiquitous

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intracellular bacterium infecting humans and a wide variety of animals. Transmission of the infection is primarily but not exclusively airborne, and ticks are usually thought to act as vectors (Duron et al., 2015). Pilloux et al. (2018) claimed that Ixodes ricinus plays a very

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minor role as a vector and reservoir of C. burnetii in Switzerland, thus supporting previous reports demonstrating the significant role of sheep and goats in the epidemiology of Q fever

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(Szymańska-Czerwińska et al., 2015; Gache et al., 2017). Farmers belong to a group with an

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increased risk of C. burnetii infection, presumably because of their contact with infected livestock (Szymańska-Czerwińska et al., 2015). Francisella tularensis is a highly virulent intracellular bacterium that may infect a

wide range of hosts, including invertebrates, mammals and birds, causing a zoonotic disease called tularemia. Humans can be infected through direct contact with infected animals, a contaminated environment or bites of infected arthropod vectors (Hestvik et al., 2014).

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Although F. tularensis infects several mammal species, antibodies against F. tularensis were also detected in wild animals, e.g. in 7 % of foxes and 1 % of wild boar (Otto et al., 2014). Nevertheless, only lagomorphs and rodents seem to have importance in the transmission of the infection (Rossow et al., 2014). Peak densities of rodent populations may trigger tularemia outbreaks. It is nevertheless unclear which small mammals are the main source of F. tularensis in Central Europe. The aim of the study was therefore to assess the prevalence of antibodies against

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C. burnetii and F. tularensis in wild small mammals.

Material and methods

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Sampling was performed using spring-loaded and live mouse traps in three Moravian localities (Poodří Protected Landscape Area, the Moravian Karst and the Mohelno National

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Natural Monument) from May to November in the years 2012 – 2015. Poodří is situated in northern Moravia (GPS: 49°69'98.23"N, 18°09'00.50"E), and trapping was carried out within

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an area of 10 ha in the Bažantula forest area, characterised by an oak Ficario-Ulmetum alnetosum association forest alternating with meadows. The Moravian Karst is situated in

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South Moravia and trapping was carried out within an area of 20 ha in the surroundings of Skalní Mlýn (GPS: 49°19'43.22"N; 16°43'23.52"E), which are characterised by beech forests

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complemented with oak and hornbeam woods and wet meadows. Mohelno is situated in

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South Moravia and trapping was performed along the Oslava River in a deep canyon valley of the Mohelno Serpentine Steppe National Nature Reserve (GPS: 49°6'10.60'' N, 16°11'21.82''E) within an area of 4 ha. Traps were placed on the ground in a line at a distance of 7 m from each other. A total of 614 wild small mammals belonging to rodents (Apodemus agrarius, A. flavicollis, A. sylvaticus, Myodes glareolus and Microtus arvalis) and insectivores (Sorex araneus and

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Talpa europaea) were trapped. Data about the animals and the localities (animal species, sex, locality and year of trapping) are summarized in Table 1. Blood drawn from the carotid artery of anaesthetised living individuals was used to obtain serum that was stored at -18°C until assayed. Animals caught by spring-loaded mouse trap were dissected and their hearts were put into 0.85% physiological solution for 1-2 days at 4°C. Thereafter, the solution was centrifuged and the drained supernatant (heart extract) was stored at -18°C until assayed. The samples (sera or heart extracts) were examined by the modified enzyme-linked

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immunosorbent assay used for detection of Borrelia burgdorferi s.l. antibodies (ELISA, TestLine, Brno, Czech Republic). The same conditions as used for ELISA plate production were used in our study with a single modification (C. burnetii and F. tularensis antigen was

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inserted instead of B. burgdorferi s.l. antigen), which has already been described in a previous study by Vostal and Žákovská (2003). Goat anti-mouse IgM and IgG conjugates (Sigma-

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Aldrich spol. s.r.o., Prague, Czech Republic) were used in the test. Sera were diluted 100x,

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heart extracts were diluted according to the protein concentration of 100x diluted sera, therefore the protein concentration was the same in both types of samples. Positive controls were prepared by immunisation of BALB/c mice with 300 μl of C. burnetii and F. tularensis

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antigens (40 μg/ml of antigen and 1 mg/ml aluminium hydroxide in 0.85% physiological solution) according to Žákovská et al. (2013). Inactivated suspensions of bacteria F. tularensis

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(Bioveta a.s., Ivanovice na Hané, Czech Republic) and C. burnetii (Batch NO. 87, Virological

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Institute Academy of Sciences, Slovakia) were used as antigens in ELISA and for immunisation of mice to obtain positive controls. Serum of wild mouse negative to C. burnetii and F. tularensis served as negative controls. Absorbance of samples was measured at 492 nm by spectrophotometer (SLT RainBow, Schoeller instruments s.r.o., Czech Republic); OD of IgM and IgG positive controls for both F. tularensis and C. burnetii had the approximate

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value of 1.0. Samples were evaluated as positive in the case of IgM or IgG, or both IgM and IgG antibodies being detected. The results were statistically analysed, taking into consideration species composition, sex, locality and year of trapping. Data analysis was performed with Pearson's chi-square test for independence using STATISTICA Cz 12 (StatSoft, Inc., 2013). We tested the null hypothesis that F. tularensis and C. burnetii seroprevalence does not differ in species, sex, locality and year of capture. The differences were considered statistically significant if the p-

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value was < 0.05. In the case of a statistically significant difference of seroprevalence in some of the variables, the Scheffé multiple comparison method (StatSoft, Inc., 2013) was subsequently applied. This method was used to identify a statistically significant difference

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between pairs of species, localities and years of trapping, by calculating the odds ratio (OR) for those pairs. Cluster analysis with the K-diameter method was used for the evaluation of

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IgM and IgG ELISA positive, dubious and negative samples. Cluster analysis was applied to

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the data plotted in sub-graphs. The data showed normal distribution based on the ShapiroWilk and Kolmogorov-Smirnov tests (StatSoft, Inc., 2013).

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Results

Antibodies against C. burnetii and F. tularensis were detected in 74 (12%) and in 41

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(7%) animals, respectively. Results according to animal species, sex, locality and year of

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trapping, are summarized in Table 1. The prevalence of antibodies against C. burnetii and F. tularensis depended on animal species and sex (p < 0.05); in the case of C. burnetii there was also a dependence on the year of collection. The prevalence of antibodies against both C. burnetii and F. tularensis differed in M. glareolus (24% and 14%, respectively, OR = 3.12, 95% CI: 1.83-5.32) and A. flavicollis (9% and 6%, respectively, OR = 2.83, 95% CI: 1.455.54). There was also a difference in the prevalence of antibodies against C. burnetii in

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M. glareolus (24 %) and A. sylvaticus (4 %) (OR = 7.32, 95% CI: 6.59-8.05). The prevalence of antibodies against C. burnetii and F. tularensis varied in females (15% and 9%, respectively, OR = 1.73, 95% CI: 1.04-2.89) and males (9% and 4%, respectively, OR = 2.37, 95% CI: 2.02-2.73). Antibodies against C. burnetii were detected in 19 % of animals caught in the year 2012, while only in 8% of animals caught in the year 2014 (OR = 2.58, 95% CI: 2.26-2.90). Coinfection with both C. burnetii and F. tularensis was found in 27 (4.4%) animals of three different species (14 M. glareolus, 11 A. flavicollis and two A. sylvaticus).

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The coinfection was higher in females than in males (23 and four, respectively), and in the Moravian Karst higher than in Poodří and Mohelno (22, four and one, respectively).

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Discussion

Blood serum is a standard type of sample to determine the presence of specific

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antibodies in humans and animals. In the case of dead rodents, heart extract was used as an

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adequate replacement for missing serum, since heart has been proved as a tissue with frequent occurrence of pathogens such as B. burgdorferi s.l. Heart extracts were adequately diluted to match the serum protein value (Grzesik et al., 2004). Before ELISA was used on wild rodents,

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the sensitivity of the commercial IgM and IgG conjugates (in antigen-free ELISA) was tested on laboratory BALB/C mice and wild rodents (M. glareolus and A. flavicollis). The

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seropositivity of laboratory mice and wild rodents was 100 % and 20 %, respectively.

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Similarly, other authors e.g. Frandsen et al. (1995) used anti-mouse conjugates to determine antibodies against B. burgdorferi in wild rodents. In the Czech Republic, there is a growing trend for the consumption of animal

products coming from small farms, so it is necessary to monitor and prevent the transmission of infections. Enserink (2010) recorded a rise of Q fever cases in the Netherlands but not knowing what triggered this explosive outbreak, which has sickened mainly people who never

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had contact with animals. To our knowledge, Q fever, remains underestimated and only partly understood. Coxiella burnetii can infect mammals, birds and arthropods, including ticks. Bacteria transmission through faeces was proven e.g. in domestic ruminants (Porter et al., 2011) and based on the data provided by Abdel-Moein and Hamza (2018), transmission through rodent faeces can not be also excluded. In the United Kingdom, antibodies against C. burnetii were detected in 17% of 796 wild rodents, ranging from 16% to 19% in different animal species, including M. glareolus,

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Microtus agrestis and A. sylvaticus (Meredith et al., 2015). In Great Britain, antibodies against C. burnetii were detected in 7% and 53% of wild brown rats and rats trapped on farms, respectively (Webster et al., 1995). In contrast, negative results were obtained in 110

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rodents (M. glareolus, A. flavicollis, A. sylvaticus and M. arvalis) from Austria, tested serologically and by RT-PCR/PCR (Schmidt et al., 2014). Prevalence obtained by direct

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detection of C. burnetii by using PCR is usually lower compared to prevalence obtained by

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using serological methods. In Italy, C. burnetii was found by PCR only in two (Apodemus spp.) of 143 (1.4%) rodents coming from the Gran Sasso e Monti della Laga National Park (Pascucci et al., 2015). No C. burnetii at all was found by PCR in 119 rodents (mainly M.

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arvalis) coming from three Q fever endemic areas in Southern Germany (Pluta et al., 2010), suggesting that these animals do not play an important role in the local epidemiology of Q

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fever however risk to humans cannot be excluded (Abdel-Moein and Hamza, 2018).

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Antibodies against C. burnetii were detected e.g. in 6.5% of 216 employees of the National Forests in Poland (Żukiewicz-Sobczak et al., 2014) and in 39% of 151 farm workers (Szymańska-Czerwińska et al., 2015). The prevalence (12%) found in wild small mammals in our study could mean that there is an increased occurrence of bacteria C. burnetii circulating in nature. However, to our knowledge, there was only one case of Q fever reported in the Czech Republic during the years 2012 - 2015 (State Health Institute data http://www.szu.cz).

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In humans in the EU, there were 777 confirmed cases of Q fever reported with a notification rate of 0.13 cases per 100 000 population (The European Union Summary Report on Trends and Sources of Zoonoses, Zoonotic Agents and Food-borne Outbreaks, 2015).

Antibodies against F. tularensis were detected in 7 % of wild small mammals. However, in Europe there are several studies, mainly based on the use of the methods of molecular biology. For example, F. tularensis was detected by PCR in 5 out of 547 (0.9 %)

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wild small mammals from Finland, with only one positive species M. agrestis (Rossow et al., 2014), and in 1.4 % of wild rodents from Italy (Pascucci et al., 2015). In Bulgaria, F. tularensis was detected by PCR in 37 of 169 (22 %) rodents. However, this was in the area

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where tularemia occurred at that time (Christova and Gladinska, 2005). Negative results were obtained by both RT-PCR/PCR and serological methods in 110 wild rodents (M. glareolus, A.

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flavicollis, A. sylvaticus and M. arvalis) trapped in the region of Lower Austria (Schmidt et

study are quite different.

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al., 2014). Although Austria is a neighboring country of the Czech Republic, results from our

In the EU, an average of patients with tularemia is 0.18 cases per 100,000 population

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(The European Union Summary Report on Trends and Sources of Zoonoses, Zoonotic Agents and Food-borne Outbreaks, 2015). The Czech Republic is among the countries with the

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highest incidence of tularemia, with an average of 43 patients with tularemia per year and

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0.45 per 100,000 inhabitants (State Health Institute data, http://www.szu.cz). In Austria, a neighboring country of the Czech Republic, no case of tularemia has been reported and its notification rate is 0 cases per 100,000 inhabitants. In contrast, the highest notification rate has been reported in Sweden (1.56 cases per 100,000 inhabitants). In our study, we found three animal species (M. glareolus, A. flavicollis and A. sylvaticus) to have antibodies against C. burnetii and F. tularensis with 4.4% coinfection.

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To our knowledge, there is no information about the coinfection of these two microorganisms in these animal species. As follows from our study, we found a statistical difference of both C. burnetii and F. tularensis prevalence in animal species, with the highest being in M. glareolus, while some other species (M. arvalis, S. araneus and T. europaea) were negative. In contrast, Meredith et al. (2015) did not find a difference in the prevalence of antibodies against C. burnetii (19, 17 and 16 %) in three tested animal species (M. agrestis, M. glareolus and A. sylvaticus, respectively). The occurrence of antibodies in wild small

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mammals suggests that their tissues may be an appropriate target for the survival of some pathogenic microorganisms. In our study, the prevalence also differed statistically in sex (antibodies against both pathogens were revealed more in females than in males). Sex

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differences must be a serious consideration in the epidemiological studies, as well as in vaccine development. This is supported by the fact that Sunagar et al. (2016) observed the

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impact of sex on vaccine efficacy against F. tularensis (enhanced levels of Ft-specific

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antibodies in serum and broncho-alveolar lavage fluid post-challenge and survival in

Conclusion

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vaccinated female mice, unlike in males).

Q fever and tularemia are mandatory reportable diseases in the Czech Republic. In the

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last ten years, there have only been five cases of Q fever, while the average number of

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tularemia cases in this period reached 59 (www.szu.cz). Due to the high prevalence of C. burnetii and F. tularensis in captured animals, risk to humans cannot be excluded.

Acknowledgements This research was supported partly by EurNegVec COST Action TD1303 and the Specific Research Programme at Masaryk University, Czech Republic.

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Ethical statement The trapping of animals and the sampling were done according to the approved experimental projects (Poodří 39/2012, 45/2013, 78/2013, 41/2015, Moravian Karst 38/2012, 6/2013, 77/2013, 42/2015) approved by the Administration of the Poodří Protected Landscape Area, by the Administration of the Moravian Karst, and by the Ministry of the Environment of

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the Czech Republic.

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Table 1: Prevalence of antibodies (both IgG and IgM) against Coxiella burnetii and Francisella tularensis tested by ELISA in wild small mammals at three localities in the Czech Republic.

Number

Positive (%)

Positive (%)

P-value 0.0001

7 (18 %) 30 (9 %) 2 (4 %) 35 (24 %) 0 0 0

336 278

49 (15 %) 25 (9 %)

25 (19 %) 11 (12 %) 19 (8 %) 19 (12 %) 74 (12 %)

0.0716

1 (3 %) 33 (8 %) 7 (4 %)

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130 95 225 164 614

0.0039

30 (9 %) 11 (4 %)

0.0754 5 (17 %) 54 (14 %) 15 (8 %)

0.0069 2 (5 %) 18 (6 %) 1 (2 %) 20 (14 %) 0 0 0

0.0342

29 392 193

P-value

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40 324 48 145 6 50 1

0.0275

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Animal species Apodemus agrarius Apodemus flavicollis Apodemus sylvaticus Myodes glareolus Microtus arvalis Sorex araneus (Talpa europaea)* Sex Females Males Locality Mohelno Moravian Karst Poodří Year 2012 2013 2014 2015 Total

Francisella tularensis

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Characteristic

Coxiella burnetii

0.1436

7 (5 %) 2 (2 %) 17(8 %) 15(9 %) 41 (7 %)

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*Talpa europaea – this species was not included in statistical evaluation according to animal species, because of the small number of animals.

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