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Francisella tularensis, the bacterium that causes the zoonosis tularemia, and its genetic near neighbor species, can be difficult or impossible to cultivate from complex samples. Thus, there is a lack of genomic information for these species that has, among other things, limited the development of robust detection assays for F. tularensis that are both specific and sensitive. The objective of this study was to develop and validate approaches to capture, enrich, sequence, and analyze Francisella DNA present in DNA extracts generated from complex samples. RNA capture probes were designed based upon the known pan genome of F. tularensis and other diverse species in the family Francisellaceae. Probes that targeted genomic regions also present in non-Francisellaceae species were excluded, and probes specific to particular Francisella species or phylogenetic clades were identified. The capture-enrichment system was then applied to diverse, complex DNA extracts containing low-level Francisella DNA, including human clinical tularemia samples, environmental samples (i.e., animal tissue and air filters), and whole ticks/tick cell lines, which was followed by sequencing of the enriched samples. Analysis of the resulting data facilitated rigorous and unambiguous confirmation of the detection of F. tularensis or other Francisella species in complex samples, identification of mixtures of different Francisella species in the same sample, analysis of gene content (e.g., known virulence and antimicrobial resistance loci), and high-resolution whole genome-based genotyping. The benefits of this capture-enrichment system include: even very low target DNA can be amplified; it is culture-independent, reducing exposure for research and/or clinical personnel and allowing genomic information to be obtained from samples that do not yield isolates; and the resulting comprehensive data not only provide robust means to confirm the presence of a target species in a sample, but also can provide data useful for source attribution, which is important from a genomic epidemiology perspective., Competing Interests: The authors have declared that no competing interests exist.

Tularaemia (rabbit fever) is a rare infection caused by Francisella tularensis, which can be transmitted from hares and rats to humans by ticks. We present two case reports of patients with tularaemia. Both were initially referred on suspicion of cancer. A 52-year-old woman, who had had a prior tick bite, was referred on suspicion of breast cancer, and a 28-year-old man was referred on suspicion of occult cancer because of fever and lymphadenopathy in the groin. Tularaemia should be considered as a differential diagnosis in cases of unexplained fever and regional lymphadenopathy, especially in patients with a history of tick bites.

To develop effective and accurate typing of strains of Francisella tularensis, a potent human pathogen and a putative bioterrorist agent, we combined analysis of insertion-deletion (indel) markers with multiple-locus variable-number tandem repeat analysis (MLVA). From 5 representative F. tularensis genome sequences, 38 indel markers with canonical properties, i.e., capable of sorting strains into major genetic groups, were selected. To avoid markers with a propensity for homoplasy, we used only those indels with 2 allelic variants and devoid of substantial sequence repeats. MLVA included sequences with much diversity in copy number of tandem repeats. The combined procedure allowed subspecies division, delineation of clades A.I and A.II of subspecies tularensis, differentiation of Japanese strains from other strains of subspecies holarctica, and high-resolution strain typing. The procedure uses limited amounts of killed bacterial preparations and, because only 1 single analytic method is needed, is time- and cost-effective.

The migratory behavior of wild birds contributes to the geographical spread of ticks and their microorganisms. In this study, we aimed to investigate the dispersal and co-occurrence of Francisella and spotted fever group Rickettsia (SFGR) in ticks infesting birds migrating northward in the African-Western Palaearctic region (AWPR). Birds were trapped with mist nests across the Mediterranean basin during the 2014 and 2015 spring migration. In total, 575 ticks were collected from 244 birds. We screened the ticks for the species Francisella tularensis, the genus Francisella, and SFGR by microfluidic real-time PCR. Confirmatory analyses and metagenomic sequencing were performed on tick samples that putatively tested positive for F. tularensis during initial screenings. Hyalomma rufipes was the most common tick species and had a high prevalence of Francisella, including co-occurrence of Francisella and SFGR. Metagenomic analysis of total DNA extracted from two H. rufipes confirmed the presence of Francisella, Rickettsia, and Midichloria. Average nucleotide identity and phylogenetic inference indicated the highest identity of the metagenome-assembled genomes to a Francisella-like endosymbiont (FLE), Rickettsia aeschlimannii, and Midichloria mitochondrii. The results of this study suggest that (i) FLE- and SFGR-containing ticks are dispersed by northbound migratory birds in the AWPR, (ii) H. rufipes likely is not involved in transmission of F. tularensis in the AWPR, and (iii) a dual endosymbiosis of FLEs and Midichloria may support some of the nutritional requirements of H. rufipes. [ABSTRACT FROM AUTHOR]

A milder climate has during the last decade contributed to an increased density and spreading of ixodid ticks, thus enhancing their role as emerging vectors for pathogenic microorganisms in northern Europe. It remains unclear if they contribute to the occurrence of infections caused by the bacteria Bartonella spp., Francisella tularensis subspecies holarctica and the parasite Toxoplasma gondii in Sweden and on the Åland islands, Finland. In this study, we want to improve understanding of the tick-borne transmission of these pathogens. Volunteers were recruited at primary healthcare centers. Ticks and blood samples were acquired from participants recruited in 2008 and 2009. Health questionnaires were completed, and medical records were acquired where applicable. Feeding time was estimated and screening of pathogens in the ticks was performed through real-time PCR. Ticks (n = 1849) were of mixed developmental stages: 76 larvae, 1295 nymphs, 426 adults and 52 undetermined. All analyzed ticks were considered negative for these pathogens since the CT-values were all below the detection limit for Bartonella spp. (1663 ticks), Francisella spp. (1849 ticks) and Toxoplasma gondii (1813 ticks). We assume that infections with these pathogens are caused by other transmission pathways within these regions of Sweden and the Åland islands, Finland. [ABSTRACT FROM AUTHOR]

Summer outbreaks of tularemia that occurred from 1995 through 2005 in 2 locations in Sweden affected 441 persons. We performed an epidemiologic investigation of these outbreaks using a novel strategy, involving high-resolution genotyping of Francisella tularensis isolates obtained from 136 patients (using 18 genetic markers developed from 6 F. tularensis genome sequences) and interviews with the patients. Strong spatial associations were found between F. tularensis subpopulations and the places of disease transmission; infection by some subpopulations occurred within areas as small as 2 km², indicating unidentified environmental point sources of tularemia. In both locations, disease clusters were associated with recreational areas beside water, and genetic subpopulations were present throughout the tularemia season and persisted over years. High-resolution genotyping in combination with patients' statements about geographic places of disease transmission provided valuable indications of likely sources of infection and the causal genotypes during these tularemia outbreaks. [ABSTRACT FROM AUTHOR]

In recent years, an increasing diversity of species has been recognized within the family Francisellaceae. Unfortunately, novel isolates are sometimes misnamed in initial publications or multiple sources propose different nomenclature for genetically highly similar isolates. Thus, unstructured and occasionally incorrect information can lead to confusion in the scientific community. Historically, detection of Francisella tularensis in environmental samples has been challenging due to the considerable and unknown genetic diversity within the family, which can result in false positive results. We have assembled a comprehensive collection of genome sequences representing most known Francisellaceae species/strains and restructured them according to a taxonomy that is based on phylogenetic structure. From this structured dataset, we identified a small number of genomic regions unique to F. tularensis that are putatively suitable for specific detection of this pathogen in environmental samples. We designed and validated specific PCR assays based on these genetic regions that can be used for the detection of F. tularensis in environmental samples, such as water and air filters. [ABSTRACT FROM AUTHOR]