Epidemiology of tularemia in the countries of the WHO Eastern Mediterranean Region (EMRO): A systematic review and meta-analysis.

BACKGROUND: Francisella tularensis, the bacterium that causes tularemia, has been a persistent and widespread pathogen in various regions of the world for centuries. Francisella tularensis can affect humans and various domestic and wild animals. The current study aimed to determine the epidemiological status of tularemia in countries of the WHO Eastern Mediterranean Region (EMRO) through a systematic review and meta-analysis. METHODS: All included studies were identified through a systematic search of online databases, including Scopus, PubMed, Web of Science, and EMBASE, through July 26, 2022, using keywords and suitable combinations. We focused on cross-sectional studies investigating the prevalence of F. tularensis. The weighted pooled prevalence was calculated using a random-effects model. RESULTS: A total of 206 studies were identified, of which 20 were finally included in the analysis. The human seroprevalence of tularemia in WHO-EMRO countries was 6.2% (95% CI, 4.2 9.2). In the subgroup analysis, anti-F. tularensis antibodies were found in 6.92% and 5.5% of the high-risk individuals and Iran, respectively. The pooled prevalence of F. tularensis in environmental samples (water and soil) from the WHO-EMRO countries was 5.8% (9.4% by PCR and 0.5% by culture). In addition, 2.5% (95% CI, 0.2 0.22.7) of ticks in WHO-EMRO countries were positive for F. tularensis. The pooled prevalence of F. tularensis in rodents is 2.0% (1.1% by PCR and 3.7% by serology). In addition, 0.6% of domestic ruminants (0.4% by PCR and 2.4% by serology) were positive for F. tularensis in WHO-EMRO countries. CONCLUSION: According to the results of the present study, tularemia is an endemic but neglected disease in the WHO-EMRO region. However, most studies on tularemia are limited to a few countries in this region. Studies on tularemia in human populations, reservoirs, and vectors have been conducted in all countries in the WHO-EMRO region to obtain more detailed information about the epidemiology of tularemia in these regions.

Targeting Tularemia: Clinical, Laboratory, and Treatment Outcomes From an 11-year Retrospective Observational Cohort in Northern Sweden.

BACKGROUND: Tularemia is an important reemerging disease with a multimodal transmission pattern. Treatment outcomes of current recommended antibiotic regimens (including ciprofloxacin and doxycycline) remain unclear. In this retrospective cohort study, we report clinical, laboratory, geographical, and treatment outcomes of laboratory-confirmed tularemia cases over an 11-year period in Northern Sweden. METHODS: Data from reported tularemia cases (aged >10 years at time of study) in Norrbotten county between 2011 and 2021 were collected through review of electronic medical records and participant questionnaires; 415 of 784 accepted participation (52.9%). Of these, 327 were laboratory-confirmed cases (serology and/or polymerase chain reaction). A multivariable logistic regression model was used to investigate variables associated with retreatment. RESULTS: Median age of participants was 54 years (interquartile range [IQR], 41.5-65) and 49.2% were female. Although ulceroglandular tularemia was the predominant form (n = 215, 65.7%), there were several cases of pulmonary tularemia (n = 40; 12.2%). Inflammatory markers were largely nonspecific, with monocytosis frequently observed (n = 36/75; 48%). Tularemia was often misdiagnosed on presentation (n = 158, 48.3%), with 65 (19.9%) receiving initial inappropriate antibiotics and 102 (31.2%) retreated. Persistent lymphadenopathy was infrequent (n = 22, 6.7%), with 10 undergoing surgical interventions. In multivariable analysis of variables associated with retreatment, we highlight differences in time until receiving appropriate antibiotics (8 [IQR, 3.25-20.75] vs 7 [IQR, 4-11.25] days; adjusted P = .076), and doxycycline-based treatment regimen (vs ciprofloxacin; adjusted P = .084), although this was not significant after correction for multiple comparisons. CONCLUSIONS: We comprehensively summarize clinical, laboratory, and treatment outcomes of type B tularemia. Targeting tularemia requires clinical awareness, early diagnosis, and timely commencement of treatment for an appropriate duration.

Detection and Genotyping of Francisella tularensis in Animal Hosts and Vectors from Six Different Natural Landscape Areas, Gansu Province, China.

OBJECTIVE: Tularemia, also known as hare fever, is caused by the bacterium Francisella tularensis (F. tularensis) transmitted through diseased wild animals, blood sucking insects, or contaminated water or food, which is distributed worldwide. The purpose of this study was to investigate F. tularensis infection in animal hosts and vectors from six different natural landscape areas in Gansu Province and to identify the genotypes of the detected F. tularensis. METHODS: Rodents were captured by snap traps, and ticks were collected by dragging a cloth over the vegetation or from domestic animals. After species identification, DNA was isolated from the captured animals and detected by nested PCR assays targeting the F. tularensis fopA gene. The positive samples were further amplified to discriminate the species, and another two short-sequence tandem repeat regions (SSTR) were amplified to identify their genotypes. All positive fragments were sequenced and analyzed by ClustalX (5.0) and DNAClub software. RESULTS: A total of 407 rodents of 12 species were captured, among which six rodent species were positive for F. tularensis, with an overall prevalence of 3.93%. The geographical difference in infection rate was statistically significant. At the SSTR9 locus, there were 7 genotypes among positive rodent samples. A total of 1864 ticks were tested for evidence of tularemia by nested PCR assays, 69 of which were positive, with an average positive rate of 3.70% for F. tularensis in ticks. The positive rates were significantly different among different regions. Seven genotypes were identified at the SSTR9 locus, one of which seemed dominant in positive tick samples. All positive samples had the same genotype at the SSTR16 locus. CONCLUSION: There is natural infection of F. tularensis among animal vectors and hosts in Gansu Province, with diverse genotypes.

Surveillance for diseases, pathogens, and toxicants of muskrat (Ondatra zibethicus) in Pennsylvania and surrounding regions.

Using diagnostic data and contemporary sampling efforts, we conducted surveillance for a diversity of pathogens, toxicants, and diseases of muskrats (Ondatra zibethicus). Between 1977 and 2019, 26 diagnostic cases were examined from Kansas and throughout the Southeast and Mid-Atlantic, USA. We identified multiple causes of mortality in muskrats, but trauma (8/26), Tyzzer's disease (5/6), and cysticercosis (5/26) were the most common. We also conducted necropsies, during November 2018-January 2019 Pennsylvania muskrat trapping season, on 380 trapper-harvested muskrat carcasses after the pelt was removed. Tissue samples and exudate were tested for presence of or exposure to a suite of pathogens and contaminants. Gastrointestinal tracts were examined for helminths. Intestinal helminths were present in 39.2% of necropsied muskrats, with Hymenolepis spp. (62%) and echinostome spp. (44%) being the most common Molecular testing identified a low prevalence of infection with Clostridium piliforme in the feces and Sarcocystis spp. in the heart. We detected a low seroprevalence to Toxoplasma gondii (1/380). No muskrats were positive for Francisella tularensis or Babesia spp. Cysticercosis was detected in 20% (5/26) of diagnostic cases and 15% (57/380) of our trapper-harvested muskrats. Toxic concentrations of arsenic, cadmium, lead, or mercury were not detected in tested liver samples. Copper, molybdenum, and zinc concentrations were detected at acceptable levels comparative to previous studies. Parasite intensity and abundance were typical of historic reports; however, younger muskrats had higher intensity of infection than older muskrats which is contradictory to what has been previously reported. A diversity of pathogens and contaminants have been reported from muskrats, but the associated disease impacts are poorly understood. Our data are consistent with historic reports and highlight the wide range of parasites, pathogens and contaminants harbored by muskrats in Pennsylvania. The data collected are a critical component in assessing overall muskrat health and serve as a basis for understanding the impacts of disease on recent muskrat population declines.

An outbreak of tularemia in southwestern Turkey.

INTRODUCTION: Tularemia has reemerged and spread throughout Turkey, and the number of cases has increased. In this study, we report on a waterborne outbreak of tularemia in the spring of 2013 in a region which was previously disease-free, and we investigated the reasons for the outbreak. METHODOLOGY: The index case, a 17-year-old male, was diagnosed with oropharyngeal tularemia. An outbreak investigation was initiated after receiving information from other patients with similar symptoms from the same village along with Balkica, Tavas, and Denizli. An epidemiological and environmental investigation was conducted. Tonsil swab specimens/lymph node aspirates collected from patients, and water samples collected from unchlorinated drinking water sources, were cultured. Additionally, a real-time polymerase chain reaction (RT-PCR) was performed on these samples. Serum samples from patients were analyzed for antibody response. RESULTS: A total of 7 patients were found in this outbreak investigation. The attack rate was found to be 1% among the people of the village and 25% among patients' family members. The drinking-water system was contaminated with F. tularensis during this outbreak. CONCLUSIONS: Lack of appropriate water infrastructure and sanitation was the primary reason for this tularemia outbreak in Turkey. Improving the water source infrastructure and sanitation should be the primary approach to preventing tularemia outbreaks.

Francisella tularensis human infections in a village of northwest Iran.

BACKGROUND: Recent seroepidemiological studies have suggested that tularemia could be an endemic bacterial zoonosis in Iran. METHODS: From January 2016 to June 2018, disease cases characterized by fever, cervical lymphadenopathy and ocular involvement were reported in Youzband Village of Kaleybar County, in the East Azerbaijan Province, northwestern Iran. Diagnostic tests included Francisella tularensis serology (including tube agglutination test and ELISA), PCR, and culture. RESULTS: Among 11 examined case-patients, the tularemia tube agglutination test was positive in ten and borderline in one. PCR detected the F. tularensis ISFtu2 elements and fopA gene in one rodent and a spring water sample from the same geographic area. CONCLUSIONS: Based on the clinical manifestations of the disease suggesting an oropharyngeal form of tularemia, serology results in case patients, and F. tularensis detection in the local fauna and aquatic environment, the water supply of the village was the likely source of the tularemia outbreak. Intervention such as dredging and chlorination of the main water storage tank of the village and training of villagers and health care workers in preventive measures and treatment of the illness helped control the infection.

High-resolution melting PCR assay as a powerful tool for the epidemiological surveillance of tularemia in Western Europe.

In Europe, tularemia is caused by Francisella tularensis subsp. holarctica and is a sporadic disease affecting mainly wildlife animals and humans. Classification of this species relies on canonical single nucleotide polymorphisms (canSNPs). Four main clades have been described for F. tularensis subsp. holarctica: B.4, B.6, B.12 and B.16. Phylogeographic studies have shown that clade B.6 is predominant in Western Europe and B.12 in Eastern and Central Europe. Based on this global phylogeny, we aimed to design a molecular typing assay for all genetic subclades of subclade B.11, which is the predominant subclade in clade B.6. We designed high-resolution melting (HRM) primers for the screening of 109 canSNPs divided in seven orders of discrimination for the molecular epidemiology analysis and tracking of Francisella tularensis subsp. holarctica in Western Europe.

Tularemia: a re-emerging tick-borne infectious disease.

Tularemia is a bacterial disease of humans, wild, and domestic animals. Francisella tularensis, which is a Gram-negative coccobacillus-shaped bacterium, is the causative agent of tularemia. Recently, an increase in the number of human tularemia cases has been noticed in several countries around the world. It has been reported mostly from North America, several Scandinavian countries, and certain Asian countries. The disease spreads through vectors such as mosquitoes, horseflies, deer flies, and ticks. Humans can acquire the disease through direct contact of sick animals, consumption of infected animals, drinking or direct contact of contaminated water, and inhalation of bacteria-loaded aerosols. Low infectious dose, aerosol route of infection, and its ability to induce fatal disease make it a potential agent of biological warfare. Tularemia leads to several clinical forms, such as glandular, ulceroglandular, oculoglandular, oropharyngeal, respiratory, and typhoidal forms. The disease is diagnosed through the use of culture, serology, or molecular methods. Quinolones, tetracyclines, or aminoglycosides are frequently used in the treatment of tularemia. No licensed vaccine is available in the prophylaxis of tularemia and this is need of the time and high-priority research area. This review mostly focuses on general features, importance, current status, and preventive measures of this disease.

Reevaluating limits of detection of 12 lateral flow immunoassays for the detection of Yersinia pestis, Francisella tularensis, and Bacillus anthracis spores using viable risk group-3 strains.

AIM: Rapid detection of biological agents in biodefense is critical for operational, tactical and strategic levels as well as for medical countermeasures. Yersinia pestis, Francisella tularensis, and Bacillus anthracis are high priority agents of biological warfare or bioterrorism and many response forces use lateral flow assays (LFAs) for their detection. Several companies produce these assays, which offer results in short time and are easy to use. Despite their importance, only few publications on the limits of detection (LOD) for LFAs are available. Most of these studies used inactivated bacteria or risk group-2 strains. As the inactivation process in previous studies might have affected the tests' performances, it was our aim in this study to determine and compare the LOD of several commercially available LFAs using viable risk group-3 strains. METHODS AND RESULTS: Lateral flow assays from four different companies for the detection of following bacteria were evaluated: Y. pestis, F. tularensis and B. anthracis spores. Two independent quantification methods for each target organism were applied, in order to ensure high quantification accuracy. LODs varied greatly between tests and organisms and ranged between 104 for Y. pestis-tests and as high as >109 for one B. anthracis-test. CONCLUSION: This work precisely determined the LODs of LFAs from four commercial suppliers. The herein determined LODs differed from results of previous studies. This illustrates the need for using accurately quantified viable risk group 3-strains for determining such LODs. SIGNIFICANCE AND IMPACT OF THE STUDY: Our work bridges an important knowledge gap with regard to LFA LOD. The LODs determined in this study will facilitate better assessment of LFA-results. They illustrate that a negative LFA result is not suited to exclude the presence of the respective agent in the analyzed sample.

Quick identification and epidemiological characterization of Francisella tularensis by MALDI-TOF mass spectrometry.

INTRODUCTION: Currently, Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) is being evaluated for its efficacy as a fast bacterial typing tool due to its great speed compared to other molecular methods. In this study, we evaluated MALDI-TOF as a tool for quick identification and typing of Francisella tularensis. MATERIALS AND METHODS: This study encompassed 86 strains from two different geographical origins (Spain and the Czech Republic), which were previously characterised by Pulsed-Field Gel Electrophoresis (PFGE) and Multiple-Locus Variable Number Tandem Repeat Analysis (MLVA). The direct colony method was used for microbial identification. High-quality spectra of the 86 strains were obtained and their main spectra profiles (MSPs) were created for epidemiological typing using MALDI-TOF. Based on the MSPs, principal components were generated and a dendrogram was constructed. An in-house MALDI-TOF library entry was created for each group of PFGE and MLVA strains based on their high-quality spectra. Two dendrograms were obtained using these entries and the unique peaks in each entry were searched. RESULTS: All strains were correctly identified to the species level. No clear divisions were found in the 86-strain dendrogram; however, Spanish and Czech strains appeared separately in dendrograms created using MLVA and PFGE entries. Entries from our in-house MALDI-TOF library revealed 2-4 biomarker peaks for the detection of the five PFGE groups and 1-12 biomarker peaks for the detection of the seven MLVA groups. Finally, two and one specific biomarkers were found in the Czech and Spanish strains, respectively. CONCLUSION: MALDI-TOF can be used to accurately identify F. tularensis strains in less than 15 min. Moreover, data on geographical origin and PFGE and MLVA groups could be obtained in less than one hour after colony growing.

VOC fingerprints: metabolomic signatures of biothreat agents with and without antibiotic resistance.

Category A and B biothreat agents are deemed to be of great concern by the US Centers for Disease Control and Prevention (CDC) and include the bacteria Francisella tularensis, Yersinia pestis, Burkholderia mallei, and Brucella species. Underscored by the impact of the 2020 SARS-CoV-2 outbreak, 2016 Zika pandemic, 2014 Ebola outbreak, 2001 anthrax letter attacks, and 1984 Rajneeshee Salmonella attacks, the threat of future epidemics/pandemics and/or terrorist/criminal use of pathogenic organisms warrants continued exploration and development of both classic and alternative methods of detecting biothreat agents. Volatile organic compounds (VOCs) comprise a large and highly diverse group of carbon-based molecules, generally related by their volatility at ambient temperature. Recently, the diagnostic potential of VOCs has been realized, as correlations between the microbial VOC metabolome and specific bacterial pathogens have been identified. Herein, we describe the use of microbial VOC profiles as fingerprints for the identification of biothreat-relevant microbes, and for differentiating between a kanamycin susceptible and resistant strain. Additionally, we demonstrate microbial VOC profiling using a rapid-throughput VOC metabolomics method we refer to as 'simultaneous multifiber headspace solid-phase microextraction' (simulti-hSPME). Finally, through VOC analysis, we illustrate a rapid non-invasive approach to the diagnosis of BALB/c mice infected with either F. tularensis SCHU S4 or Y. pestis CO92.

Selective Predation by Owls on Infected Bank Voles (Myodes glareolus) as a Possible Sentinel of Tularemia Outbreaks.

Tularemia is a widely spread zoonotic disease in the northern hemisphere, caused by the bacterium Francisella tularensis. In humans, tularemia is an acute febrile illness with incidence peaks in late summer to early autumn of outbreak years, but there is no early warning system in place that can reduce the impact of disease by providing timely risk information. In this study, we revisit previously unpublished data on F. tularensis in water, sediment, soil, and small mammals from 1984 in northern Sweden. In addition, we used human case data from the national surveillance system for tularemia in the same year. In the environmental and small mammal material, bank vole (Myodes glareolus) samples from urine and bladder were the only samples that tested positive for F. tularensis. The prevalence of F. tularensis among trapped bank voles was 13.5%, although all six bank voles that were retrieved from owl nest boxes in early May tested positive. Forty-two human tularemia cases were reported from August to December in 1984. Based on these results, we encourage investigating the potential role of tularemia-infected bank voles retrieved from owl nest boxes in spring as an early warning for outbreaks of tularemia among humans in summer and autumn of the same year.

[A severely ill 33-year-old woman with hepatic abscesses]. / Schweres Krankheitsgefühl und Leberabszesse bei einer 33­jährigen Patientin.

A 33-year-old woman in a seriously ill state presented with hepatic abscesses. The proof of epitheloid-like reactions by biopsy and further serological analysis led to the final diagnosis of tularemia, which represents a rare disease in Germany. Thereafter targeted antibiotic therapy was successfully initiated. The contribution of simultaneously diagnosed celiac disease to the unusual manifestation of tularemia in the liver, remains uncertain.

Comprehensive Laboratory Evaluation of a Specific Lateral Flow Assay for the Presumptive Identification of Francisella tularensis in Suspicious White Powders and Aerosol Samples.

We conducted a comprehensive, multi-phase laboratory evaluation of the Tularemia BioThreat Alert® (BTA) test, a lateral flow assay (LFA) for the rapid detection of Francisella tularensis. The study, conducted at 2 sites, evaluated the limit of detection (LOD) of this assay using the virulent SchuS4 strain and the avirulent LVS strain of F. tularensis. In 6-phase evaluation (linear dynamic range and reproducibility, inclusivity, near-neighbor, environmental background, white powder, and environmental filter extract), 13 diverse strains of F. tularensis, 8 Francisella near neighbors, 61 environmental background organisms, 26 white powders, and a pooled aerosol extract were tested. In the 937 tests performed, the Tularemia BTA demonstrated an LOD of 107 to 108 cfu/mL, with a sensitivity of 100.00%, specificity of 98.08%, and accuracy of 98.84%. These performance data are important for accurate interpretation of qualitative results arising from screening suspicious white powders in the field.

Early detection of viable Francisella tularensis in environmental matrices by culture-based PCR.

BACKGROUND: Francisella tularensis is a fastidious, Gram-negative coccobacillus and is the causative agent of tularemia. To assess viability yet overcome lengthy incubation periods, a culture-based PCR method was used to detect early growth of the lowest possible number of F. tularensis cells. This method utilized a previously developed enhanced F. tularensis growth medium and is based on the change in PCR cycle threshold at the start and end of each incubation. RESULTS: To test method robustness, a virulent Type A1 (Schu4) and B (IN99) strain and the avirulent Live Vaccine Strain (LVS) were incubated with inactivated target cells, humic acid, drinking and well water, and test dust at targeted starting concentrations of 1, 10, and 100 CFU mL- 1 (low, mid, and high, respectively). After 48 h, LVS growth was detected at all targeted concentrations in the presence of 106 inactivated LVS cells; while Schu4 and IN99 growth was detected in the presence of 104 Schu4 or IN99 inactivated cells at the mid and high targets. Early detection of F. tularensis growth was strain and concentration dependent in the presence of fast-growing well water and test dust organisms. In contrast, growth was detected at each targeted concentration by 24 h in humic acid and drinking water for all strains. CONCLUSIONS: Results indicated that the culture-based PCR assay is quick, sensitive, and specific while still utilizing growth as a measure of pathogen viability. This method can circumvent lengthy incubations required for Francisella identification, especially when swift answers are needed during epidemiological investigations, remediation efforts, and decontamination verification.

Surveillance of Francisella tularensis in surface water of Kurdistan province, west of Iran.

BACKGROUND: The etiologic agent of tularemia, Francisella tularensis, is transmitted to humans via ingestion of contaminated water or food, arthropods bite, respiratory aerosols, or direct contact with infected animals body fluids or tissues. In the current study, due to the importance of water in transmitting the disease and the report of the disease in different regions of Iran, surface water of Kurdistan province were evaluated for the presence of F.tularensis. MATERIALS AND METHODS: Sampling was carried out in five-counties of Kurdistan province. Sixty-six specimens of surface water were collected. The detection was carried out by targeting ISFtu2 and fopA genes using TaqMan real-time PCR. Moreover, the samples were both cultured and inoculated into NMRI inbreed mice. Spleens of inoculated mice and bacterial isolates were tested by TaqMan real-time PCR. RESULTS: Despite the lack of isolation of F. tularensis, the results of the molecular testing indicate the presence of bacteria in surface water. Molecular positivity of one sample (1.51%) was confirmed using a real-time PCR for both ISFtu2 and fopA genes. Moreover, 4.54% of the samples were positive for ISFtu2. CONCLUSION: Since the in vitro isolation of bacteria from environmental samples is associated with a very low success rate and depends on various environmental parameters, the use of molecular techniques for monitoring of the bacteria in the contaminated areas is fully recommended.

A rare cause of granulomatous hepatitis: Tularemia.

Tularemia is a zoonotic infection caused by Francisella tularensis. Tularemia has several clinical form in humans, including ulceroglandular, pneumonic, oropharyngeal, oculoglandular, and systemic (typhoidal). Tularemia may develop granulomatous and suppurative lesions, especially in the affected regional lymph nodes and various organs. Patients with hepatic involvement typically have elevated transaminase levels, hepatomegaly and rarely jaundice. Histologically, there are typically suppurative microabscesses with occasional surrounding macrophages. Rarely, hepatic granuloma can develop due to tularemia. We present a case of an 8 year-old male residing in a rural village in Turkey, who came to our hospital after having intermittent fever for four months and right upper abdominal pain for two months. Liver had a nodular appearance in liver imaging and liver biopsy were consistent with granulomatous hepatitis. The microagglutination test was positive for tularemia in the patient who was investigated for granulomatous hepatitis etiology. Symptoms and signs improved with tularemia treatment. We present a rare case of hepatic involvement of tularemia in a child. Clinicians should be suspicious of and evaluate for typhoidal tularemia in patients who present with prolonged fever and non-specific systemic symptoms, potentially with associated abdominal pain.

Tularemia in a Prosthetic Joint Infection.

Periprosthetic infections occur in approximately 0.8% to 1.9% of all total knee arthroplasties (TKAs). Even with these low rates, it is rare to find a zoonotic bacterium causing a periprosthetic infection. In this case report, the authors identify the second documented case of a total joint infection with Francisella tularensis in the world and the first in the United States. A 58-year-old man underwent a left TKA in 1994 and a right TKA in 1997 for severe primary bilateral knee osteoarthrosis. In 2015, he underwent polyethylene exchange for polyethylene wear. Subsequently, he developed repeated effusion without fever or constitutional signs of infection. One aspiration was sent for culture and grew F tularensis. He was treated with doxycycline for chronic suppression and currently has no signs of infection. Total joint implantation rates are expected to rise, with 3.5 million procedures projected to be performed annually by the year 2030 vs 450,000 procedures performed in 2005. With the increased number of operations, it is likely that zoonotic infections will increase as well. Thus, rare zoonotic bacterial infections as well as chronic outdoor exposure in the presence of persistent joint swelling should be considered when obtaining a patient history. [Orthopedics. 2020; 43(1):e54-e56.].

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

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 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 prevalence of antibodies against C. burnetii and F. tularensis was detected in the case of M. glareolus (24 % and 14 %, respectively).