Umeå University's logo

Gram-negative bacteria utilize type VI secretion systems (T6SS) for microbial competition and host interaction. While most pathogens rely on the canonical T6SSi, Francisella species uniquely possess T6SSii. The highly virulent human pathogen Francisella tularensis harbors a distinct T6SSii variant that includes pdpC, encoding a putative effector protein. Bioinformatic analysis revealed a conserved amino acid triad in PdpC, homologous to motifs found in Make Caterpillars Floppy family toxins. To investigate the functional relevance of this triad, site-directed mutagenesis was performed in the live vaccine strain of F. tularensis, substituting each residue with alanine. Mutants showed impaired phagosomal escape, reduced intracellular replication, and marked attenuation in the mouse infection model. Equivalent mutations introduced into F. novicida, a model for T6SS-mediated secretion, confirmed the triad’s importance. Mass spectrometry analysis demonstrated that PdpC is secreted in a T6SS-dependent manner. Importantly, the mutations did not affect secretion, and deletion of pdpC did not alter the overall secretion profile. These findings indicate that the conserved triad is essential for PdpC’s effector function but dispensable for its secretion. This study highlights a critical motif required for Francisella virulence and provides new insights into the specialized mechanisms of T6SSii effectors.

Autophagy is a crucial mechanism in the host response to intracellular bacterial pathogens during which microorganisms may undergo direct degradation in autophagolysosomes. As a highly virulent intracellular pathogen, Francisella tularensis has developed survival strategies to escape from the phagosome, replicate in the cytosol of mononuclear cells, and avoid degradation within the double-membrane vacuole during the autophagy-mediated response. The aim of this study was to investigate the role of the ATG5 autophagy protein in the host immune response to Francisella tularensis subsp. holarctica, live vaccine strain (LVS), since ATG5 plays an important role in autophagosome formation during canonical autophagy. In vitro experiments were conducted on immortalized bone marrow macrophages subjected to starvation-induced autophagy. Transgenic mice deficient in ATG5 of cells of the myeloid lineage (monocytes/macrophages and granulocytes) were used to analyze the immunological responses after intradermal infection. Cytokine levels were analyzed using Luminex, RT-qPCR, and ELISA, while inflammatory cell infiltration in the lung was analyzed by immunohistochemistry. Our results demonstrate that induced autophagy decreased bacterial replication in vitro. However, ATG5 deficiency in myeloid cells in vivo significantly diminished levels of pro-inflammatory cytokine IFN-γ in the sera, spleen, liver, and lung during Francisella infection. The attenuated pro-inflammatory response also led to significantly reduced macrophage and T cell infiltration in the lung tissue. Our findings also reveal that neutralization of IL-1β in myeloid ATG5ΔMye mice increased susceptibility to tularemia by increasing bacterial burden in organs.

Vaccination with the live vaccine strain (LVS) or natural infection with Francisella tularensis confers long-lasting protection against re-infection, and it is almost exclusively dependent on cell-mediated immunity. The extent, if any, to which vaccine-induced and infection-induced immunity differ in quality and persistence remains unclear. In this study, we compared human immune responses resulting from LVS vaccination and tularemia infection. Peripheral blood mononuclear cells from vaccinees and convalescent patients were recall-stimulated and analyzed for proliferation, characterization of cytokine-producing memory T cells, and cytokine secretion. Both vaccination and infection elicited robust antigen-specific T-cell activation, accompanied by coordinated cytokine release. Intracellular staining identified multifunctional CD4 and CD8 T cells after both vaccination and infection. Characterization of memory subsets revealed that vaccination primarily induced transitional and effector memory populations, while natural infection generated broader and more persistent responses, including activation of terminally differentiated TEMRA (Terminal Effector Memory T cells re-expressing CD45RA) cells and higher intracellular expression of MIP-1β by CD4 and CD8 cells. These findings demonstrate that both LVS vaccination and natural infection induce efficacious immune responses to F. tularensis, as evidenced by the presence of long-lasting, multifunctional T cells, but with partially distinct memory profiles. The broader and more durable responses after natural infection highlight areas of relevance to understand the efficacy of existing and future tularemia vaccines.

Background: Tularemia is a re-emerging disease in Sweden, frequently affecting working-age individuals and often resulting in prolonged recovery times. The disease-related economic impact has not previously been investigated outside bioterrorism scenarios. In this study we assess the economic burden associated with endemic tularemia in Sweden.  

Method: Data on primary care visits, hospital admissions, and sick leaves were collected from participants with serology-confirmed tularemia through questionnaires and electronic medical records in Northern Sweden from 2011 to 2021. The dataset was enhanced with national cost-of-care data for tularemia in primary and specialist care from NordDRG (Diagnosis Related Group; 2021-2023), and sickness benefit data from the Social Insurance Agency (2011-2023). Total direct and indirect costs were estimated by integrating these data sources and adjusted for inflation to 2025 levels. Average salary and labor productivity (Gross Domestic Product [GDP] per person employed) was assumed.

Results: Among participants (n=294), the mean age was 52 years; 68.1% were employed or job-seeking, with 71.3% of these reporting sick leave during illness. Healthcare costs were primarily driven by general practitioner visits (mean 1.74 visits; 5,125 SEK per participant [p.p.]) and hospital admissions (15.6% of participants; mean 4.7 days; 11,268 SEK p.p.) with relatively low complexity (mean DRG weight 0.8), while antibiotic treatment and diagnostics were less costly (figure 1). Indirect costs included sick pay (≤14 days; 70.0%; 4,106 SEK p.p.), sickness benefit (>14 days; 30.0%; 5,835 SEK p.p.), and lost GDP-based productivity, and made up 75.7% of total costs (78,503 SEK p.p.).

A mean of 394 (range 87-1,048) tularemia cases per year were reported to the Swedish Public Health Agency between 2011-2023. The estimated annual societal cost of human tularemia infection was 30.9 million SEK (range 6.8-82.3 million SEK). 

Conclusion: Tularemia imposes a significant socioeconomic burden on society primarily through morbidity and prolonged recovery. Regional outbreaks could have detrimental effects on local economy and public services. Further evaluation of the cost-effectiveness of primary and secondary preventive measures is required. 

Since the 1912 discovery of Francisella tularensis as the causative microbe, tularemia has waxed and waned as a disease of public health importance. F. tularensis infects an enormous variety of animals that transmit disease to humans by a wide variety of routes, leading to several forms of human disease. Person-to-person transmission of tularemia has never been observed. Nonetheless, the extraordinary infectious potential of this intracellular bacterium made it ripe for development as a bioterrorism weapon. Tularemia can be challenging to diagnose, and vaccines are not available in most countries; fortunately, the disease is treatable with appropriate antibiotics.

Licensed vaccines against Francisella tularensis, a public health threat in some parts of the world and a potential bioterrorism agent, are lacking in Western countries. Existing tularemia vaccine candidates have not been promising in protecting against the most serious respiratory form of tularemia infection. Previous studies identified a novel live attenuated vaccine candidate, F. tularensis subsp. tularensis ΔclpB, that protected rodents against aerosol challenge with the most virulent biotype of Francisella. Characterization demonstrated that ΔclpB is amenable to modern manufacturing. Here, we evaluated further ΔclpB's protective capacities in Fischer 344 rats and in cynomolgus macaques. Results demonstrated that rats immunized intradermally with ΔclpB survived aerosol F. tularensis challenge with up to 100 median lethal doses administered one year after vaccination, accompanied by reduced clinical signs of infection as well as reduced histopathology and bacterial burdens in lungs and spleens. Moreover, intradermal ΔclpB vaccination protected macaques against at least 500 MLD of aerosol F. tularensis challenge administered one and three months after vaccination; vaccination ameliorated symptoms, bacterial burdens, and tissue pathology when tested one year after vaccination. Given the low incidence of tularemia in nature, these studies therefore lay the foundation for additional animal-based evaluations of efficacy and future safety evaluation of ΔclpB by clinical studies.

Introduction: For a majority of tularemia patients, serology is the basis for the diagnosis. The aim of this study was to perform an analysis of the samples analyzed at a Swedish reference laboratory for the presence of Francisella tularensis-specific antibody levels in sera from individuals with suspected tularemia. Annual and monthly variations of the total number of samples and proportions of positive samples were analyzed, as well as the influence of age and gender.

Methods: We performed a retrospective analysis of the presence of F. tularensis-specific antibodies in serological samples from patients with suspected tularemia analyzed during the period 2010 - 2022 at the University Hospital of Umeå in Sweden, a national reference laboratory, by use of various statistical methods. In total, some 15,100 serum samples had been analyzed for the presence of IgG and IgM antibodies by ELISA during the 13-year period.

Results: Overall, there were higher number of samples with IgG positive or borderline titers, 2,522 and 921, respectively, than with IgM positive or borderline titers, 1,802 and 409, respectively. Repeated samples were obtained from some 1,930 individuals and approximately a third of the cases, which were initially seronegative, had seroconverted when resampled. Peak number of monthly samples were recorded in August and September, > 3,000. Annual numbers varied greatly and peak numbers were observed in 2015 and 2019, 1,832 and 2,250, respectively, whereas some other years the numbers were 700 – 800. There was also much variation in the annual and monthly percentages of positive samples and they varied between less than 10% to greater than 20%. The highest percentages of positive samples were recorded in September and October. IgG and IgM titers declined with age and these differences were highly significant for IgG titers, with decreasing average titers for each 20-year interval.

Discussion: Collectively, the data demonstrate the marked annual and seasonal variations in tularemia sampling occurring in Sweden. Also, the proportion of positive samples increased during months and years with peak number of samples. Another notable finding was that average antibody titers decreased with increased age.

Background: Tularaemia is a geographically widespread disease affecting animals and humans. In Sweden, transmission patterns are complex, occurring mainly through mosquito vectors. We investigated human exposure and whether passive tularaemia surveillance (reported by the public) of deceased wild hares could be used to temporally and geographically predict outbreaks among humans. 

Methods: A survey was sent to the 830 cases of reported tularaemia in Norrbotten county, Sweden, between 2011-2021; and 313/415 (75.4%) respondents with laboratory-evidence of tularaemia were included. Geographic data from human infections in 2019 (n=54) and 2020 (n=77) was compared to data on deceased forest hares from the Swedish Veterinary Agency, matched by year and region.

Results: Respondents (n=313) rarely reported direct exposure to hares (8,6%) and/or other rodents (3.8%) during the 2-weeks prior to illness; while recreational activities (forest hiking 61.6%; mushroom/berry-picking 24.0%; fishing 11.5%; and hunting 3.8%) were more common. Peak incidence of reported deceased hares in 2019 and 2020 (n=84 and n=66; 11/15 [73.3%] and 19/21 [90.4%] PCR-positive for tularaemia, respectively) corresponded to peak incidence of symptom onset of human cases (median difference +6 days [2019] and -2 days [2020]; p=0.066 and p=0.695, respectively). Distribution of reported hares corresponded with municipalities with highest incidence of human tularaemia and location of self-reported suspected infection (Figure 1). Most reported their location of infection to be within their residential municipality (n=92/106, 86.8%).

Conclusion: Passive surveillance of tularaemia using deceased hares correlates with symptom onset in humans and could predict geographical outbreaks in the community. Surveillance of other affected/reservoir species should be considered. 

Background: Tularemia is an important re-emerging 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 2011-2021 were collected through review of electronic medical records and participant questionnaires; with 415 out of 784 accepting participation (52.9%). Of these, 327 were laboratory-confirmed cases (serology and/or PCR). A multivariable logistic regression model was used to investigate variables associated with re-treatment.

Results: Median age of participants was 54 years (IQR 41.5-65) and 49.2% were female. While 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 non-specific, with monocytosis frequently observed (n=36/75; 48%). Tularemia was often misdiagnosed upon presentation (n=158, 48.3%), with 65 (19.9%) receiving initial inappropriate antibiotics, and 102 (31.2%) re-treated. Persistent lymphadenopathy was infrequent (n=22, 6.7%), with 10 undergoing surgical interventions. In multivariable analysis of variables associated with re-treatment, we highlight differences in time until receiving appropriate antibiotics (8 [IQR 3.25-20.75] vs. 7 [IQR 4-11.25] days; adjusted p=0.076), and doxycycline-based treatment regimen (vs. ciprofloxacin; adjusted p=0.084), although not significant after correction for multiple comparisons.

Conclusion: 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.