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Tularemia is caused by subspecies of Francisella tularensis and can manifest in a variety of disease states, with the pneumonic presentation resulting in the greatest mortality. Despite decades of research, there are no approved vaccines against F. tularensis in the United States. Traditional vaccination strategies, such as live-attenuated or subunit vaccines, are not favorable due to inadequate protection or safety concerns. Because of this, novel vaccination strategies are needed to combat tularemia. Here we discuss the current state of and challenges to the tularemia vaccine field and suggest novel vaccine approaches going forward that might be better suited for protecting against F. tularensis infection. [ABSTRACT FROM AUTHOR]

Adenopathies are a frequent cause of recourse in internal medicine. When histological analysis reveals the presence of granuloma, multiple infectious or non-infectious etiologies are considered. If diagnoses of lymphoma, sarcoidosis or tuberculosis are easily mentioned, tularemia should also be considered in the differential diagnosis. A 54-year-old patient had a fever at the evening with night sweats and a cough resistant to two lines of antibiotics. A thoraco-abdomino-pelvic CT scan revealed hilar and mediastinal adenopathies that appeared hypermetabolic with PET-TDM, as well as pulmonary nodules. A PCR performed on lymph node biopsy and serology allowed the diagnosis of tularemia. The evolution was favourable after antibiotic treatment. The association of fever, night sweats, altered general state and mediastinal adenopathies should be considered as a diagnosis of tularemia. Ganglionic biopsy, combined with molecular biology techniques and serology, can confirm the diagnosis. [ABSTRACT FROM AUTHOR]

• Tularemia can be transmitted through the ingestion of contaminated grape products. • F. tularensis can be introduced into the food chain via mechanical harvesting of grapes and presumably infected rodents. • The clinical course of tularemia can be unexpectedly long and require long antibiotic treatment durations. Tularemia is caused by Francisella tularensis and can occasionally establish foodborne transmission. Patients were identified by active case detection through contact with the treating physicians and consent for publication was obtained. Clinical data were accumulated through a review of the patient charts. Serology, culture, and PCR methods were performed for confirmation of the diagnosis. A 46-year-old patient was hospitalised in the University Hospital Frankfurt (a tertiary care hospital) for pharyngitis and cervical lymphadenitis with abscess. A diagnosis of tularemia was made serologically, but treatment with ciprofloxacin initially failed. F. tularensis was detected in pus from the lymph node using a specific real-time PCR. The use of RD1 PCR led to the identification of the subspecies holarctica. Antibiotic therapy with high-dose ciprofloxacin and gentamicin was administered and was subsequently changed to ciprofloxacin and rifampicin. During a must-tasting, five other individuals became infected with tularemia by ingestion of contaminated must. All patients required treatment durations of more than 14 days. Mechanically harvested agricultural products, such as wine must, can be a source of infection, probably due to contamination with animal carcasses. The clinical course of tularemia can be complicated and prolonged and requires differentiated antibiotic treatment. [ABSTRACT FROM AUTHOR]

Abstract Alveolar macrophages resident in the lung are prominent phagocytic effector cells of the pulmonary innate immune response, and paradoxically, are attractive harbors for pathogens. Consequently, facultative intracellular bacteria, such as Francisella tularensis , can cause severe systemic disease and sepsis, with high morbidity and mortality associated with pulmonary infection. Current clinical treatment, which involves exhaustive oral or intravenous antibiotic therapy, has limitations such as systemic toxicity and off-target effects. Pulmonary administration represents a promising alternative to systemic dosing for delivering antibiotics directly to the lung. Here, we present synthesized mannosylated ciprofloxacin polymeric prodrugs for efficient pulmonary delivery, targeting, and subsequent internalization by alveolar macrophages. We demonstrate significant improvement in efficacy against intracellular infections in an otherwise uniformly lethal airborne Francisella murine model (F. novicida). When administered to the lungs of mice in a prophylactic regimen, the mannosylated ciprofloxacin polymeric prodrugs led to 50% survival. In a treatment regimen that was concurrent with infection, the survival of mice increased to 87.5%. Free ciprofloxacin antibiotic was ineffective in both cases. This significant difference in antibacterial efficacy demonstrates the impact of this delivery platform based on improved physiochemical, pharmacokinetic, and pharmacodynamic properties of ciprofloxacin administered via our glycan polymeric prodrug. This modular platform provides a route for overcoming the limitations of free drug and increasing efficacy in treatment of intracellular infection. Graphical abstract Image 1 [ABSTRACT FROM AUTHOR]

A β-class carbonic anhydrase (CA, EC 4.2.1.1) from the pathogenic bacterium Francisella tularensis (FtuβCA) was cloned and purified, and the anion inhibition profile was investigated. Based on the measured kinetic parameters for the enzyme catalyzed CO 2 hydration reaction (k cat of 9.8 × 10 5 s −1 and a k cat /K M of 8.9 × 10 7 M −1 s −1 ), FtuβCA is a highly effective enzyme. The activity of FtuβCA was not inhibited by a range of anions that do not typically coordinate Zn(II) effectively, including perchlorate, tetrafluoroborate, and hexafluorophosphate. Surprisingly, some anions which generally complex well with many cations, including Zn(II), also did not effectively inhibit FtuβCA, e.g., fluoride, cyanide, azide, nitrite, bisulphite, sulfate, tellurate, perrhenate, perrhuthenate, and peroxydisulfate. However, the most effective inhibitors were in the range of 90–94 µM (sulfamide, sulfamic acid, phenylarsonic and phenylboronic acid). N,N -Diethyldithiocarbamate (K I of 0.31 mM) was a moderately potent inhibitor. As Francisella tularensis is the causative agent of tularemia, the discovery of compounds that can interfere with the life cycle of this pathogen may result in novel opportunities to fight antibiotic drug resistance. [ABSTRACT FROM AUTHOR]

Background Tularemia is caused by Francisella tularensis , a gram-negative bacterium that has been weaponized as an aerosol. For protection of personnel conducting biodefense research, the United States Army required clinical evaluation of a new lot of tularemia live vaccine strain manufactured in accordance with Current Good Manufacturing Practices. Methods A phase 2 randomized clinical trial compared the new lot (DVC-LVS) to the existing vaccine that has been in use for decades (USAMRIID-LVS). The vaccines were delivered by scarification to 228 participants. Safety, reactogenicity, take and/or antibody levels were assessed on days 0, 1, 2, 8, 14, 28, 56, and 180. Principal Results Both vaccines were safe and had acceptable reactogenicity profiles during six months of follow-up. There were no serious or grade 3 and 4 laboratory adverse events. Moderate systemic reactogenicity (mostly headache or feeling tired) was reported by ∼23% of participants receiving either vaccine. Injection site reactogenicity was mostly mild itchiness and pain. The frequencies of vaccine take skin reactions were 73% (95% CI, 64, 81) for DVC-LVS and 80% (95% CI, 71, 87) for USAMRIID-LVS. The 90% CI for the difference in proportions was −6.9% (−16.4, 2.6). The rates of seroconversion measured by microagglutination assay on days 28 or 56 were 94% (95% CI, 88, 98; n = 98/104) for DVC-LVS and 94% (95% CI, 87, 97; n = 103/110) for USAMRIID-LVS (p = 1.00). Day 14 sera revealed more rapid seroconversion for DVC-LVS relative to USAMRIID-LVS: 82% (95% CI, 73, 89) versus 55% (95% CI, 45, 65), respectively (p < 0.0001). Major conclusions The DVC-LVS vaccine had similar safety, reactogenicity, take and antibody responses compared to the older USAMRIID vaccine, and was superior for early (day 14) antibody production. Vaccination take was not a sensitive surrogate for seroconversion in a multi-center study where personnel at five research clinics performed assessments. ClinicalTrials.gov identifier NCT01150695 [ABSTRACT FROM AUTHOR]

Tularemia or rabbit fever is a transmissible disease from animals, rodents, and insects to human populations that is caused by Francisella tularensis. Epidemiological studies showed that tularemia is endemic throughout most different regions of the world. Recent evidence documented the transmission of the F. tularensis in a different part of Asia. Because there is no updated review information for tularemia in Iran, we performed this systematic review. In this study, we systematically explored biomedical databases (Google Scholar, Scopus, PubMed, and Web of sciences) to identify epidemiology, reservoirs, and carriers of Francisella in animal and human clinical specimens from 2010 to 2020, either in English or in Persian. Different studies have shown the different frequencies of F. tularensis among human and animal resources in eighteen provinces of Iran. In total, 1242 human clinical specimens, 1565 animal samples, and 355 environmental water samples were investigated to find F. tularensis in different provinces of Iran. According to the collected documents, 94 human clinical samples, 69 water samples, and 26 animal specimens were introduced as positive samples for the F. tularensis. According to studies, thirteen species of rodent and hare presented as an inter-epizootic reservoir. Only one species of tick (D. marginatus) was introduced as a vector for Francisella in Iran. According to these results, it is essential for exclusive attention to the prevalence of F. tularensis in different provinces of Iran. Furthermore, special planning should be done for prevention, control of the outbreak, and proper treatment of the tularemia. [ABSTRACT FROM AUTHOR]

A new β-class carbonic anhydrase (CA, EC 4.2.1.1) has been cloned, purified and characterized in the genome of the pathogenic bacterium Francisella tularensis responsible of the febrile illness tularemia. This enzyme, FtuβCA, showed a k cat of 9.8 × 10 5 s −1 and a k cat /K M of 8.9 × 10 7 M −1 s −1 for the CO 2 hydration, physiological reaction, being one of the most effective β-CAs known to date, with a catalytic activity only 1.68-times lower than that of the human(h) isoform hCA II. A panel of 39 simple aromatic and heterocyclic sulfonamides, as well as clinically used drugs incorporating sulfonamide/sulfamate zinc-binding groups, was used to investigate the inhibition profile of FtuβCA with these classes of derivatives. The enzyme generally showed a weaker affinity for these inhibitors compared to other α- and β-CAs investigated earlier, with only acetazolamide and its deacetylated precursor having inhibition constant <1 µM. Indeed, the two compounds acetazolamide AAZ and its deacetylated precursor 13 (K I s of 655–770 nM), as well as metanilamide and methazolamide (K I s of 2.53–2.92 µM), were the best FtuβCA inhibitors detected so far. As the physiological role of bacterial β-CAs is poorly understood for the virulence/life cycle of these pathogens, the present study may constitute a starting point for the design of effective pathogenic bacteria CA inhibitors with potential use as antiinfectives. [ABSTRACT FROM AUTHOR]

Francisella tularensis subsp. tularensis strain SchuS4 (Ftt) is a highly virulent intracellular bacterium. Inhalation of 10 or fewer organisms results in an acute and potentially lethal disease called pneumonic tularemia. Ftt infections occur naturally in the U.S. and Ftt was developed as a bioweapon. Thus, there is a need for vaccines that protect against this deadly pathogen. Although a live vaccine strain of Francisella tularensis (LVS) exists, LVS fails to generate long-lived protective immunity against modest challenge doses of Ftt. We recently identified an important role for high avidity CD4 + T cells in short-term protection and hypothesized that expanding this pool of cells would improve overall vaccine efficacy with regard to longevity and challenge dose. In support of our hypothesis, application of a prime/boost vaccination strategy increased the pool of high avidity CD4 + T cells which correlated with improved survival following challenge with either increased doses of virulent Ftt or at late time points after vaccination. In summary, we demonstrate that both epitope selection and vaccination strategies that expand antigen-specific T cells correlate with superior immunity to Ftt as measured by survival. [ABSTRACT FROM AUTHOR]

Francisella tularensis is the cause of the zoonotic disease tularemia. In Sweden and Scandinavia, epidemiological studies have implicated mosquitoes as a vector. Prior research has demonstrated the presence of Francisella DNA in infected mosquitoes but has not shown definitive transmission of tularemia from a mosquito to a mammalian host. We hypothesized that antimicrobial peptides, an important component of the innate immune system of higher organisms, may play a role in mosquito host-defense to Francisella . We established that Francisella sp. are susceptible to two cecropin antimicrobial peptides derived from the mosquito Aedes albopictus as well as Culex pipiens . We also demonstrated induced expression of Aedes albopictus antimicrobial peptide genes by Francisella infection C6/36 mosquito cell line. We demonstrate that mosquito antimicrobial peptides act against Francisella by disrupting the cellular membrane of the bacteria. Thus, it is possible that antimicrobial peptides may play a role in the inability of mosquitoes to establish an effective natural transmission of tularemia. [ABSTRACT FROM AUTHOR]

Tularemia is a zoonotic disease that has been reported in many countries of the Northern Hemisphere. However, in some countries, such as Iran, this disease has been neglected by the health care system, and it is under-reported. Here we report an unusual case of ulceroglandular tularemia occurring in a 35-year-old woman who presented with a skin lesion of the left flank, inguinal lymphadenopathy, and an abdominal abscess. The serological and real-time PCR tests for tularemia were positive for this patient, and infection by Francisella tularensis subsp. holarctica was confirmed. It is necessary to implement various educational programs to increase the awareness of physicians with tularemia. [ABSTRACT FROM AUTHOR]

Francisella tularensis ( Ft ) is a Category A biothreat agent for which there currently is no FDA-approved vaccine. Thus, there is a substantial effort underway to develop an effective tularemia vaccine. While it is well established that gender can significantly impact susceptibility to primary infection, the impact of gender on vaccine efficacy is not well established. Thus, development of a successful vaccine against tularemia will require an understanding of the impact gender has on vaccine-induced protection against this organism. In this study, a role for gender in vaccine-induced protection following Ft challenge is identified for the first time. In the present study, mucosal vaccination with inactivated Ft (i Ft ) LVS elicited gender-based protection in C57BL/6Tac mice against respiratory challenge with Ft LVS. Specifically, vaccinated male mice were more susceptible to subsequent Ft LVS challenge. This increased susceptibility in male mice correlated with increased bacterial burden, increased tissue inflammation, and increased proinflammatory cytokine production late in post-challenge infection. In contrast, improved survival of i Ft -vaccinated female mice correlated with reduced bacterial burden and enhanced levels of Ft -specific Abs in serum and broncho-alveolar lavage (BAL) fluid post-challenge. Furthermore, vaccination with a live attenuated vaccine consisting of an Ft LVS superoxide dismutase (SodB) mutant, which has proven efficacious against the highly virulent Ft SchuS4 strain, demonstrated similar gender bias in protection post- Ft SchuS4 challenge. Of particular significance is the fact that these are the first studies to demonstrate that gender differences impact disease outcome in the case of lethal respiratory tularemia following mucosal vaccination. In addition, these studies further emphasize the fact that gender differences must be a serious consideration in any future tularemia vaccine development studies. [ABSTRACT FROM AUTHOR]

Tularaemia is a zoonotic disease caused by Francisella tularensis, a Gram-negative, facultative intracellular bacterium. Typically, human and animal infections are caused by F tularensis subspecies tularensis (type A) strains mainly in Canada and USA, and F tularensis subspecies holarctica (type B) strains throughout the northern hemisphere, including Europe. In the past, the epidemiological, clinical, therapeutic, and prognostic aspects of tularaemia reported in the English medical literature were mainly those that had been reported in the USA, where the disease was first described. Tularaemia has markedly changed in the past decade, and a large number of studies have provided novel data for the disease characteristics in Europe. In this Review we aim to emphasise the specific and variable aspects of tularaemia in different European countries. In particular, two natural lifecycles of F tularensis have been described in this continent, although not fully characterised, which are associated with different modes of transmission, clinical features, and public health burdens of tularaemia. [ABSTRACT FROM AUTHOR]

Francisella tularensis is a human zoonotic pathogen and the causative agent of tularemia, a severe infectious disease. Given the extreme infectivity of F. tularensis and its potential to be used as a biological warfare agent, a fast and sensitive detection method is highly desirable. Herein, we construct a novel detection platform composed of two units: (1) Magnetic beads conjugated with multiple capturing antibodies against F. tularensis for its simple and rapid separation and (2) Genetically-engineered apoferritin protein constructs conjugated with multiple quantum dots and a detection antibody against F. tularensis for the amplification of signal. We demonstrate a 10-fold increase in the sensitivity relative to traditional lateral flow devices that utilize enzyme-based detection methods. We ultimately envision the use of our novel nanoprobe detection platform in future applications that require the highly-sensitive on-site detection of high-risk pathogens. [ABSTRACT FROM AUTHOR]

Tularemia is a zoonotic disease caused by Francisella tularensis , which is transmitted to humans most commonly by contact with infected animals, tick bites, or inhalation of aerosolized bacteria. F. tularensis is highly infectious via the aerosol route; inhalation of as few as 10–50 organisms can cause pneumonic tularemia. Left untreated, the pneumonic form has more than >30% case-fatality rate but with early antibiotic intervention can be reduced to 3%. This study compared tularemia disease progression across three species of nonhuman primates [African green monkey (AGM), cynomolgus macaque (CM), and rhesus macaque (RM)] following aerosolized F. tularensis Schu S4 exposure. Groups of the animals exposed to various challenge doses were observed for clinical signs of infection and blood samples were analyzed to characterize the disease pathogenesis. Whereas the AGMs and CMs succumbed to disease following challenge doses of 40 and 32 colony forming units (CFU), respectively, the RM lethal dose was 276,667 CFU. Following all challenge doses that caused disease, the NHPs experienced weight loss, bacteremia, fever as early as 4 days post exposure, and tissue burden. Necrotizing-to-pyogranulomatous lesions were observed most commonly in the lung, lymph nodes, spleen, and bone marrow. Overall, the CM model consistently manifested pathological responses similar to those resulting from inhalation of F. tularensis in humans and thereby most closely emulates human tularemia disease. The RM model displayed a higher tolerance to infection and survived exposures of up to 15,593 CFU of aerosolized F. tularensis . [ABSTRACT FROM AUTHOR]

Abstract: Tularemia is a highly infectious zoonotic disease caused by a Gram-negative coccoid rod bacterium, Francisella tularensis. Tularemia is considered as a life-threatening potential biological warfare agent due to its high virulence, transmission, mortality and simplicity of cultivation. In the work reported here, different electrochemical immunosensor formats for the detection of whole F. tularensis bacteria were developed and their performance compared. An anti-Francisella antibody (FB11) was used for the detection that recognises the lipopolysaccharide found in the outer membrane of the bacteria. In the first approach, gold-supported self-assembled monolayers of a carboxyl terminated bipodal alkanethiol were used to covalently cross-link with the FB11 antibody. In an alternative second approach F(ab) fragments of the FB11 antibody were generated and directly chemisorbed onto the gold electrode surface. The second approach resulted in an increased capture efficiency and higher sensitivity. Detection limits of 4.5ng/mL for the lipopolysaccharide antigen and 31 bacteria/mL for the F. tularensis bacteria were achieved. Having demonstrated the functionality of the immunosensor, an electrode array was functionalised with the antibody fragment and integrated with microfluidics and housed in a tester set-up that facilitated complete automation of the assay. The only end-user intervention is sample addition, requiring less than one-minute hands-on time. The use of the automated microfluidic set-up not only required much lower reagent volumes but also the required incubation time was considerably reduced and a notable increase of 3-fold in assay sensitivity was achieved with a total assay time from sample addition to read-out of less than 20min. [Copyright &y& Elsevier]

Francisella tularensis is the causative agent of tularemia an infectious zoonotic disease. We attempted the molecular detecting of F. tularensis in small ruminants and ticks attached to these animals in Kurdistan province (the west of Iran). In this study, 250 blood and 244 tick samples were collected from sheep and goats and were tested for F. tularensis IS Ftu2 gene detection using the Real Time-TaqMan PCR method. The collected ticks were morphologically classified as Dermacentor marginatus (67.2%), Rhipicephalus turanicus (12.30%), Rhipicephalus sanguineus (10.66%), and Haemaphysalis concinna (9.83%). No positive F. tularensis were identified in animal blood samples. F. tularensis was detected in 2 (0.82%) ticks samples. Positive samples were identified as F. tularensis subsp. holarctica and collected from D. marginatus ticks and Divandareh county. In this study, the presence of F. tularensis in ticks of Kurdistan province was confirmed, the possible role of ticks in the transmission to livestock and human through tick bites in this region should be considered. • F. tularensis was detected in 0.82% of small ruminant ticks in western Iran. • F. tularensis was detected in Dermacentor marginatus ticks. • Positive samples were identified as F. tularensis subsp. holarctica. • F. tularensis could be endemic in Iran. [ABSTRACT FROM AUTHOR]

Due to the high infectivity and fatal effect on human population, Francisella tularensis (F. tularensis) is classified as a potential biological warfare agent. The interaction between host and pathogen behind the successful establishment of F. tularensis infection within the human host is largely unknown. In our present work, we have studied the molecular level interactions between the host cellular components and F. tularensis genes to understand the interplay between the host and pathogen. Interestingly, we have identified the pathways associated with the pathogen offensive strategies that help in invasion of host defensive systems. The F. tularensis genes purL, katG, proS, rpoB and fusA have displayed high number of interactions with the host genes and thus play a crucial role in vital pathogen pathways. The pathways identified were involved in adaptation to different stress conditions within the host and might be crucial for designing new therapeutic interventions against tularemia. A total number of 33 whole genome sequences available for various F. tularensis strains were downloaded from NCBI database. The phylogenetic tree was constructed using ParSNP tool and visualized using iTOL tool. The representative strain F. tularensis SUH4 was highlighted in yellow color. (A) Phylogenetic tree in rooted tree layout, and (B) Circular tree view of phylogenetic tree. [Display omitted] • Phylogenetic tree of 33 whole genome sequences of Francisella strains constructed. • Gene interaction network between 377 host and 237 pathogen genes was constructed. • Clustering analysis of gene networks resulted densely inter connected gene clusters. • FEA has resulted with key pathways which play critical role in host and pathogen. • Important pathogen genes were identified and reported as new potential drug targets. [ABSTRACT FROM AUTHOR]

Highlights: [•] IFN-? is required for survival during secondary pneumonic tularemia. [•] IL-17A is dispensable for survival during secondary pneumonic tularemia. [•] Vaccination with LVS clpB elicits the same memory T cell response as LVS. [ABSTRACT FROM AUTHOR]

Summary: Background: The first human case of tularemia in Iran was reported in 1980 and there have been no subsequent reports of tularemia in the country. The aim of this study was to carry out a survey of tularemia among different groups in the province of Kurdistan in western Iran. Methods: The following information was collected by means of an in-house questionnaire: participant demographic characteristics, exposure to risks, and use of appropriate personal protective equipment and disinfectant in their occupation. A blood sample was collected from each participant. Sera were tested using an ELISA kit (Virion\Serion) to detect specific IgG antibodies against Francisella tularensis. Results: Of a total of 250 serum samples, 14.40% had anti-tularemia IgG antibodies. The highest seroprevalence was found in hunters (18%) and the lowest in health care workers (12%). Age had a significant positive association with tularemia seroprevalence (p <0.001). The seroprevalence of tularemia in people exposed to foxes (hunting or eating the meat) (25%) was significantly higher than in others (8.65%) (p = 0.01). Conclusions: According to the findings of this study, it is highly recommended that physicians and health care workers are informed about bacteria circulating in this area. By sensitizing the health system, it is expected that some cases of the clinical disease will be reported in the near future. Similar studies in other parts of the country and on domestic and wild animals will clarify the epidemiology of tularemia in Iran. [Copyright &y& Elsevier]

Summary: Background: Over the last two to three decades there has been a marked decrease in certain bacterial infections in Turkey. One of them is tuberculosis. Of note, statistics published by the Turkish Ministry of Health (MoH) show decreasing pulmonary tuberculosis (PTB), but on the other hand, increasing extrapulmonary tuberculosis (EPTB). The most common form of EPTB is tuberculous cervical lymphadenitis (TCL). The increase in the number of TCL cases despite the decline in cases of PTB is seen as a paradoxical issue. In contrast there has been an increase in the number of oropharyngeal tularemia cases in the last decade in Turkey. The aim of this study was to draw attention to the importance of differentiating between TCL and tularemia lymphadenitis, because these diseases may have a similar histopathological appearance. Methods: Thirty-two cases diagnosed as TCL were identified from the archives of a pathology laboratory (Patomer Pathology Laboratory, Bursa, Turkey). PCR tests for Francisella tularensis and Mycobacterium tuberculosis were carried out on the paraffin tissue blocks of these cases. At the same time, statistical data on PTB and EPTB for the period 1996–2010 were retrieved from the MoH and reviewed. Statistics related to tularemia, which has been diagnosed since 1988 and has been increasing in the last 10 years, were obtained from the Department of Zoonoses of the MoH. Results: Six out of 32 (19%) cases who had previously been diagnosed with ‘casseifying granulomatous lymphadenitis consistent with tuberculosis’ were found to be positive for tularemia by PCR test of the cervical lymph nodes. Conclusions: Oropharyngeal tularemia should be kept in mind in the differential diagnosis of cervical lymphadenitis in widespread geographic regions of the Northern Hemisphere, including the Asian continent. In particular, if granulomatous inflammation with necrosis is seen histopathologically, tularemia should be excluded before making the diagnosis of TCL. [ABSTRACT FROM AUTHOR]

Abstract: Francisella tularensis is a highly infectious facultative intracellular bacterium causing the zoonotic disease tularemia. Numerous attributes required for F. tularensis intracellular multiplication have been identified recently. However, the mechanisms by which the majority of them interfere with the infected host are still poorly understood. The following review summarizes our current knowledge on the different steps of Francisella intramacrophagic life cycle and expands on the importance of nutrient acquisition. [Copyright &y& Elsevier]

Francisella tularensis is an important human pathogen responsible for causing tularemia. F. tularensis has long been developed as a biological weapon and is now classified as a category A agent by the Centers for Disease Control because of its possible use as a bioterror agent. F. tularensis represses inflammasome; a cytosolic multi-protein complex that activates caspase-1 to produce proinflammatory cytokines IL-1β and IL-18. However, the Francisella factors and the mechanisms through which F. tularensis mediates these suppressive effects remain relatively unknown. Utilizing amutant of F. tularensis in FTL_0325 gene, this study investigated the mechanisms of inflammasome repression by F. tularensis. We demonstrate that muted IL-1 and IL-18 responses generated in macrophages infected with F. tularensis live vaccine strain (LVS) or the virulent SchuS4 strain are due to a predominant suppressive effect on TLR2-dependent signal 1. Our results also demonstrate that FTL_0325 of F. tularensis impacts proIL-1β expression as early as 2 h post-infection and delays activation of AIM2 and NLRP3-inflammasomes in a TLR2-dependent fashion. An enhanced activation of caspase-1 and IL-1β observed in FTL_0325 mutant-infected macrophages at 24 h post-infection was independent of both AIM2 and NLRP3. Furthermore, F. tularensis LVS delayed pyroptotic cell death of the infected macrophages in an FTL_0325-dependent manner during the early stages of infection. In vivo studies in mice revealed that suppression of IL-1β by FTL_0325 early during infection facilitates the establishment of a fulminate infection by F. tularensis. Collectively, this study provides evidence that F. tularensis LVS represses inflammasome activation and that F. tularensis-encoded FTL_0325 mediates this effect. [ABSTRACT FROM AUTHOR]

Highlights: [•] Interest in vaccine development against bioterror agents has increased since 2001. [•] One such agent of interest is Francisella tularensis. [•] Live attenuated strains have shown promise as vaccine candidates in mouse models. [•] Survival and protection data from recent examples is reviewed in this article. [Copyright &y& Elsevier]

B Methods and materials: b Two searchers were conducted using: «tularemia outbreak» and «francisella tularensis outbreak» in title and/or abstract using PubMed database for articles that had been published until 01.10.2018. B Background: b Tularemia is a highly contagious, multi-systemic zoonotic disease caused by francisella tularensis that can give rise to outbreaks. Tularemia outbreaks can occur via several transmission ways; evidence shows that even though a certain group, for example one family, is infected with the same source, individuals can develop different forms of the disease according to the port of entry. [Extracted from the article]

Abstract: Francisella tularensis, the causative agent of tularemia, is commonly transmitted by ticks. To ensure accurate F. tularensis reporting rates in epidemiological surveys, specific discrimination between F. tularensis and Francisella-like tick endosymbionts (FLEs) is absolutely critical. Four molecular available techniques capable of distinguishing Francisella spp. were compared here for the first time in French Dermacentor reticulatus ticks in order to estimate their specificity as well as their ease and speed of use. Results showed that tul4 and fopA real-time PCR assays can easily and effectively discriminate between F. tularensis and FLEs in D. reticulatus. In addition, a high prevalence of FLEs in D. reticulatus collected in France was reported by the use of fopA real-time PCR assay (79%). Finally, phylogenetic analysis showed that FLEs isolated from D. reticulatus correspond to a well-defined group compared to FLEs originating from various tick species. [Copyright &y& Elsevier]

Abstract: Francisella tularensis (F. tularensis), the causative agent of tularemia, has long been known to invade and occupy non-phagocytic epithelial cells. Many epithelial cell infection models have been developed to study this process; however, due to the lack of consensus on infection methods and precise experimental procedures to evaluate invasion and replication, selection of appropriate models to use based on the literature is challenging. To evaluate in vitro non-phagocytic cell infection models, we chose 8 epithelial cultured cell lines from published models to infect with F. tularensis subspecies novicida (F. novicida) and compared the results to a recently developed model that used the mouse hepatocyte BNL CL.2 cell line. We utilized classical gentamicin-based invasion assays to determine total intracellular bacterial loads and employed microscopic examination with staining techniques that distinguished between intracellular and extracellular bacteria to provide an accurate assessment of the proportion of invaded host cells and the degree of bacterial replication. We found that COS-7 cells exhibited the greatest invasion rates; CMT-93 cells contained the largest intracellular bacterial loads; ad HEK-293s were capable of invasion and replication rates at high levels, but required shorter infection incubation times. Although COS-7, CMT-93 and HEK-293 cell lines may be suited to study certain aspects of invasion or replication, we found that BNL CL.2 cells appeared the most appropriate to study the overall pathogenesis of F. novicida when examined in toto. [Copyright &y& Elsevier]

Pneumonic tularemia is caused by inhalation of Francisella tularensis, one of the most infectious microbes known. We wanted to study the kinetics of the initial and early interactions between bacterium and host cells in the lung. To do this, we examined the infection of A549 airway epithelial cells with the live vaccine strain (LVS) of F. tularensis. A549 cells were infected and analyzed for global transcriptional response at multiple time points up to 16 h following infection. At 15 min and 2 h, a strong transcriptional response was observed including cytoskeletal rearrangement, intracellular transport, and interferon signaling. However, at later time points (6 and 16 h), very little differential gene expression was observed, indicating a general suppression of the host response consistent with other reported cell lines and murine tissues. Genes for macropinocytosis and actin/cytoskeleton rearrangement were highly up-regulated and common to the 15 min and 2 h time points, suggesting the use of this method for bacterial entry into cells. We demonstrate macropinocytosis through the uptake of FITC-dextran and amiloride inhibition of Francisella LVS uptake. Our results suggest that macropinocytosis is a potential mechanism of intracellular entry by LVS and that the host cell response is suppressed during the first 2-6 h of infection. These results suggest that the attenuated Francisella LVS induces significant host cell signaling at very early time points after the bacteria's interaction with the cell. [ABSTRACT FROM AUTHOR]

Both tuberculosis cervical lymphadenitis ( TCL) and oropharyngeal tularaemia ( OT) have similar signs, symptoms and pathological findings. We aimed to investigate the frequency of tularaemia antibodies in patients diagnosed with TCL. Using data from the Tuberculosis Control Dispensaries between the years of 2008 and 2011 in Turkey, all patients diagnosed with TCL were informed about and included in the study. Control group subjects were selected from healthy blood donors who lived in the same region. After informed consent was obtained, the sera obtained from volunteer TCL patients and the control group were tested with a microagglutination technique for Francisella tularensis. Antibodies to Brucella were also investigated with a tube agglutination test for cross-reactivity in sera that were seropositive for tularaemia. Sera were obtained from a total of 1170 individuals in the TCL group and 596 in the control group from 67 of 81 provinces in Turkey. Francisella tularensis-positive antibodies were found in 79 (6.75%) cases in the TCL group and two (0.33%) cases in the control group with a titre of ≥1:80 (p < 0.01). When the presence of antibody of any titre was considered, the ratio became 8.2% (96/1170) in the TCL group and 0.67% (4/596) in the control group (p < 0.001). For the first time, with this study, tularaemia serology was found to be positive in a significant portion (6.75%) of diagnosed cases of TCL. In tularaemia endemic regions, it was concluded that tularaemia serology should be investigated in patients suspected of having TCL. [ABSTRACT FROM AUTHOR]

Abstract: Tularemia is an infection caused by Francisella tularensis with a worldwide distribution in the northern hemisphere and diverse clinical manifestations. Serology plays an important role in the diagnosis of tularemia. A commercially available immunochromatographic assay (ICA) for the serologic diagnosis of tularemia (VIRapid Tularemia, Vircell, Granada, Spain) was evaluated, and the performance was compared with that of the current standard, the microagglutination test (MA). A panel of 221 sera from 109 cases of tularemia was tested as well as 236 sera from normal individuals or individuals with other infectious or autoimmune diseases. The ICA demonstrated 91.5% (κ = 0.91) agreement with the reference method (MA) and gave an overall sensitivity of 99.3% and a specificity of 94.6%. No cross-reactivity was observed in the ICA with serum samples from normal individuals and patients with autoimmune diseases and bacterial, viral, and parasitic infections, although 4 of 50 patients with brucellosis demonstrated positive results in the ICA. The performance of ICA was simple, and it requires no specialized equipment. The ease of use and significantly high sensitivity and specificity of ICA make it a good choice for diagnostic testing and a valuable field test to support a presumptive diagnosis of tularemia in remote areas. [Copyright &y& Elsevier]

Francisella tularensis, the causative agent of tularemia, is one of the deadliest agents of biological warfare and bioterrorism. Extremely high virulence of this bacterium is associated with its ability to dampen or subvert host innate immune response. The objectives of this study were to identify factors and understand the mechanisms of host innate immune evasion by F. tularensis. We identified and explored the pathogenic role of a mutant interrupted at gene locus FTL_0325, which encodes an OmpAlike protein. Our results establish a pathogenic role of FTL_0325 and its ortholog FTT0831c in the virulent F. tularensis SchuS4 strain in intramacrophage survival and suppression of proinflammatory cytokine responses. This study provides mechanistic evidence that the suppressive effects on innate immune responses are due specifically to these proteins and that FTL_0325 and FTT0831c mediate immune subversion by interfering with NF-κB signaling. Furthermore, FTT0831c inhibits NF-κB activity primarily by preventing the nuclear translocation of p65 subunit. Collectively, this study reports a novel F. tularensis factor that is required for innate immune subversion caused by this deadly bacterium. [ABSTRACT FROM AUTHOR]

Abstract: Francisella tularensis, the causative agent of tularemia, is a highly infectious intracellular pathogen with no licensed vaccine available today. The recent search for genome sequences involved in F. tularensis virulence mechanisms led to the identification of the 30-kb region defined as a Francisella pathogenicity island (FPI). In our previous iTRAQ study we described the concerted upregulation of some FPI proteins in different F. tularensis strains cultivated under stress conditions. Among them we identified the IglH protein whose role in Francisella virulence has not been characterized yet. In this work we deleted the iglH gene in a European clinical isolate of F. tularensis subsp. holarctica FSC200. We showed that the iglH gene is necessary for intracellular growth and escape of F. tularensis from phagosomes. We also showed that the iglH mutant is avirulent in a mouse model of infection and persists in the organs for about three weeks after infection. Importantly, mice vaccinated by infection with the iglH mutant were protected against subcutaneous challenge with the fully virulent parental FSC200 strain. This is the first report of a defined subsp. holarctica FPI deletion strain that provides protective immunity against subsequent subcutaneous challenge with a virulent isolate of F. tularensis subsp. holarctica. [Copyright &y& Elsevier]

Abstract: The study of membrane proteins and membrane protein complexes (MPC) provides crucial information in the field of bacterial physiology and pathogenesis research. The method of blue native polyacrylamide gel electrophoresis and its combination with SDS-PAGE (BN/SDS-PAGE) were here employed to study the membrane complexome of an intracellular bacterium Francisella tularensis, the causative agent of a severe disease tularemia. In the presented study we describe the subunit composition and stoichiometry of several MPC involved in various cell functions (oxidative phosphorylation, membrane transport, cell division, membrane or periplasmic proteins folding, iron storage, phospholipid and cell envelope biosynthesis). Moreover, some undocumented or hypothetical MPC with possible connection to virulence factors were also proposed and some newly detected subunits were assigned to known complexes. The BN/SDS-PAGE combined with mass spectrometry appeared to be a strong tool in the investigation of membrane proteins and complexes and thus successfully complements the traditional electrophoresis approaches. [Copyright &y& Elsevier]

Abstract: Francisella tularensis is a highly pathogenic gram negative bacterium that infects multiple sites in a host, including the skin and the respiratory tract, which can lead to the onset of a deadly disease with a 50% mortality rate. The live vaccine strain (LVS) of F. tularensis, while attenuated in humans but still virulent in mice, is not an option for vaccine use in the United States due to safety concerns, and currently no FDA approved vaccine exists. The purpose of the present work was to assess the ability of recombinant Francisella outer membrane protein A (FopA) to induce a protective response in mice. The gene encoding FopA from F. tularensis LVS was cloned and expressed in Escherichia coli. The resulting recombinant protein was affinity-purified from the E. coli outer membrane, incorporated into liposomes and administered to mice via multiple routes. FopA-immunized mice produced FopA-specific antibodies and were protected against both lethal intradermal and intranasal challenges with F. tularensis LVS. The vaccinated mice had reduced bacterial numbers in their lungs, livers and spleens during infection, and complete bacterial clearance was observed by day 28 post infection. Passive transfer of FopA-immune serum protected naïve mice against lethal F. tularensis LVS challenge, showing that humoral immunity played an important role in vaccine efficacy. FopA-immunization was unable to protect against challenge with the fully virulent SchuS4 strain of F. tularensis; however, the findings demonstrate proof of principle that an immune response generated against a component of a subunit vaccine is protective against lethal respiratory and intradermal tularemia. [Copyright &y& Elsevier]

Abstract: Objective: We aimed to attract our colleagues’ attention to the oropharyngeal tularemia, which has been an infrequently diagnosed disease in otolaryngology clinics also becoming widespread in Turkey. Methods: A retrospective review of database of 3 patients was performed to find patients with Tularemia. The medical files, treatment charts, serological and PCR findings and radiological (magnetic resonance imaging), histopathological records of these patients were reviewed. Results: The study group consisted of 3 oropharyngeal tularemia patients. All of them presented with tonsillopharyngitis and cervical lymphadenitis and all were resistant to beta lactam or cephalosporin group antibiotics. Two patients had magnetic resonance imaging findings. All patients had positive serological tests. Strongly positive serological tests led to the diagnosis of tularemia. All patients had positive PCR and direct fluorescent antibody (DFA) test. All patients responded to systemic streptomycin treatment. Conclusion: In the daily practice of an otolaryngologist, it is not usual to diagnose a patient with oropharyngeal tularemia. Tularemia should be considered in the differential diagnosis of massive adenotonsillar enlargement and extensive necrotic cervical lymphadenopathy not responding to beta lactam antibiotics as a result of the rising number of tularemia outbreaks outside the classic endemic areas. [Copyright &y& Elsevier]

Abstract: This study evaluated commercial automated and manual DNA extraction methods for the isolation of Francisella tularensis DNA suitable for real-time polymerase chain reaction (PCR) analysis from cell suspensions and spiked cotton, foam, and polyester swabs. Two automated methods, the MagNA Pure Compact and the QIAcube, were compared to 4 manual methods, the IT 1-2-3 DNA sample purification kit, the MasterPure Complete DNA and RNA purification kit, the QIAamp DNA blood mini kit, and the UltraClean Microbial DNA isolation kit. The methods were compared using 6 F. tularensis strains representing the 2 subspecies which cause the majority of reported cases of tularemia in humans. Cell viability testing of the DNA extracts showed that all 6 extraction methods efficiently inactivated F. tularensis at concentrations of ≤106 CFU/mL. Real-time PCR analysis using a multitarget 5′ nuclease assay for F. tularensis revealed that the PCR sensitivity was equivalent using DNA extracted by the 2 automated methods and the manual MasterPure and QIAamp methods. These 4 methods resulted in significantly better levels of detection from bacterial suspensions and performed equivalently for spiked swab samples than the remaining 2. This study identifies optimal DNA extraction methods for processing swab specimens for the subsequent detection of F. tularensis DNA using real-time PCR assays. Furthermore, the results provide diagnostic laboratories with the option to select from 2 automated DNA extraction methods as suitable alternatives to manual methods for the isolation of DNA from F. tularensis. [Copyright &y& Elsevier]

Abstract: Tularemia is a rare zoonotic disease caused by Francisella tularensis, a Gram-negative bacteria. The clinical manifestations of pulmonary tularemia resemble those of other airways infections. Recently, a case of pulmonary tularemia was diagnosed at Tufts Medical Center. The purpose of the current report is to document the utility of applying several diagnostic tools, including immunohistochemistry, electron microscopy, microbiology and molecular biology in confirming the diagnosis of pulmonary tularemia, particularly in convalescing cases (up to 3 weeks postpresentation) and after antibiotic therapy. Our study demonstrates the usefulness of microbiological studies followed by morphological evaluation and the limitation of the molecular biology analysis of posttherapy samples. [Copyright &y& Elsevier]

Abstract: Francisella tularensis, the etiological agent of tularemia, is regarded as a potential bioterrorism agent. The advent of bioterrorism has heightened awareness of the need for validated methods for processing environmental samples. In this study we determined the optimal method for processing environmental swabs for the recovery and subsequent detection of F. tularensis by the use of real-time PCR assays. Four swab processing recovery methods were compared: heat, sonication, vortexing, and the Swab Extraction Tube System (SETS). These methods were evaluated using cotton, foam, polyester and rayon swabs spiked with six pathogenic strains of F. tularensis. Real-time PCR analysis using a multi-target 5′nuclease assay for F. tularensis showed that the use of the SETS method resulted in the best limit of detection when evaluated using multiple strains of F. tularensis. We demonstrated also that the efficiency of F. tularensis recovery from swab specimens was not equivalent for all swab processing methodologies and, thus, that this variable can affect real-time PCR assay sensitivity. The effectiveness of the SETS method was independent of the automated DNA extraction method and real-time PCR platforms used. In conclusion, diagnostic laboratories can now potentially incorporate the SETS method into specimen processing protocols for the rapid and efficient detection of F. tularensis by real-time PCR during laboratory bioterrorism-related investigations. [Copyright &y& Elsevier]

Abstract: Protection against the intracellular bacterium Francisella tularensis within weeks of vaccination is thought to involve both cellular and humoral immune responses. However, the relative roles for cellular and humoral immunity in long lived protection against virulent F. tularensis are not well established. Here, we dissected the correlates of immunity to pulmonary infection with virulent F. tularensis strain SchuS4 in mice challenged 30 and 90 days after subcutaneous vaccination with LVS. Regardless of the time of challenge, LVS vaccination protected approximately 90% of SchuS4 infected animals. Surprisingly, control of bacterial replication in the lung during the first 7 days of infection was not required for survival of SchuS4 infection in vaccinated mice. Control and survival of virulent F. tularensis strain SchuS4 infection within 30 days of vaccination was associated with high titers of SchuS4 agglutinating antibodies, and IFN-γ production by multiple cell types in both the lung and spleen. In contrast, survival of SchuS4 infection 90 days after vaccination was correlated only with IFN-γ producing splenocytes and activated T cells in the spleen. Together these data demonstrate that functional agglutinating antibodies and strong mucosal immunity are correlated with early control of pulmonary infections with virulent F. tularensis. However, early mucosal immunity may not be required to survive F. tularensis infection. Instead, survival of SchuS4 infection at extended time points after immunization was only associated with production of IFN-γ and activation of T cells in peripheral organs. [ABSTRACT FROM AUTHOR]

Abstract: Francisella tularensis is a highly infectious human pathogen that can cause tularemia, but little is known about the lipid composition in membranes of this bacterium. Our recent study has shown that lipid A in membranes of F. tularensis subsp. novicida is related to its infection ability [X. Wang, A.A. Ribeiro, Z. Guan, S.N. Abraham, C.R. Raetz, Attenuated virulence of a Francisella mutant lacking the lipid A 4′-phosphatase, Proc. Natl. Acad. Sci. U.S.A. 104 (2007) 4136–4141.]. Here we purified the glycerophospholipids of F. novicida, and analyzed by using thin-layer chromatography, electrospray ionization mass spectrometry and gas chromatography/mass spectrometry. Phosphatidylglycerol, phosphatidylethanolamine, cardiolipin, phosphatidylcholine and lyso-phosphatidylethanolamine were found in F. novicida. These glycerophospholipids contain fatty acids with broad range of chain lengths, and acyl chains have unusually different length in a single glycerophospholipid molecule. The special fatty acyl composition of glycerophospholipids in F. tularensis could potentially be used to detect the bacteria. [Copyright &y& Elsevier]

Abstract: Francisella tularensis is a category A select agent. J5dLPS/OMP is a novel vaccine construct consisting of detoxified, O-polysaccharide side chain-deficient, lipopolysaccharide non-covalently complexed with the outer membrane protein of N. meningitidis group B. Immunization elicits high-titer polyclonal antibodies specific for the highly-conserved epitopes expressed within the glycolipid core that constitutes gram-negative bacteria (e.g., F. tularensis). Mice immunized intranasally with J5dLPS/OMP exhibited protective immunity to intratracheal challenge with the live vaccine strain, as well as the highly-virulent SchuS4 strain, of F. tularensis. The efficacy of J5dLPS/OMP vaccine suggests its potential utility in immunizing the general population against several different gram-negative select agents concurrently. [Copyright &y& Elsevier]

Acid phosphatase activity in the highly infectious intracellular pathogen Francisella tularensis is directly related with the ability of these bacteria to survive inside host cells. Pharmacological inactivation of acid phosphatases could potentially help in the treatment of tularemia or even be utilized to neutralize the infection. In the present work, we report inhibitory compounds for three of the four major acid phosphatases produced by F. tularensis SCHU4: AcpA, AcpB, and AcpC. The inhibitors were identified using a catalytic screen from a library of chemicals approved for use in humans. The best results were obtained against AcpA. The two compounds identified, ascorbate (Ki = 380 ± 160 μM) and 2-phosphoascorbate (Ki = 3.2 ± 0.85 μM) inhibit AcpA in a noncompetitive, nonreversible fashion. A potential ascorbylation site in the proximity of the catalytic pocket of AcpA was identified using site-directed mutagenesis. The effects of the inhibitors identified in vitro were evaluated using bioassays determining the ability of F. tularensis to survive inside infected cells. The presence of ascorbate or 2-phosphoascorbate impaired the intramacrophage survival of F. tularensis in an AcpA-dependent manner as it was probed using knockout strains. The evidence presented herein indicated that ascorbate could be a good alternative to be used clinically to improve treatments against tularemia. [ABSTRACT FROM AUTHOR]

Abstract: Aging is a complex phenomenon that has been shown to affect many organ systems including the innate and adaptive immune systems. The current study was designed to examine the potential effect of immunosenescence on the pulmonary immune response using a Francisella tularensis live vaccine strain (LVS) inhalation infection model. F. tularensis is a Gram-negative intracellular pathogen that can cause a severe pneumonia. In this study both young (8–12week old) and aged (20–24month old) mice were infected intranasally with LVS. Lung tissues from young and aged mice were used to assess pathology, recruitment of immune cell types and cytokine expression levels at various times post infection. Bacterial burdens were also assessed. Interestingly, the lungs of aged animals harbored fewer organisms at early time points of infection (day 1, day 3) compared with their younger counterparts. In addition, only aged animals displayed small perivascular aggregates at these early time points that appeared mostly mononuclear in nature. However, the kinetics of infiltrating polymorphonuclear neutrophils (PMNs) and increased cytokine levels measured in the bronchial alveolar lavage fluid (BALF) were delayed in infected aged animals relative to young infected animals with neutrophils appearing at day 5 post infection (PI) in the aged animals as opposed to day 3 PI in the young infected animals. Also evident were alterations in the ratios of mononuclear to PMNs at distinct post infection times. The above evidence indicates that aged mice elicit an altered immune response in the lung to respiratory F. tularensis LVS infections compared to their younger counterparts. [Copyright &y& Elsevier]

Francisella tularensis is a highly infectious Gram-negative bacterium causing the zoonotic disease tularemia. This facultative intracellular pathogen multiplies in vivo mainly inside macrophages, but has the capacity to infect and survive in many other cell types, including other phagocytic and nonphagocytic cells. In vitro, F. tularensis escapes rapidly from the phagosomal compartment and replicates in the cytoplasm of infected cells. An impressive number of novel genes related to F. tularensis pathogenesis have been identified recently. However, the information on biological functions still remains limited to a few of them. In this review, we will try to provide a comprehensive overview of the bacterial attributes, currently known—or suspected—to participate in F. tularensis virulence and will highlight the future challenges in F. tularensis research. [Copyright &y& Elsevier]

Francisella tularensis induces apoptosis within macrophages but the temporal and spatial modulation through activation of caspase-1, caspase-3, and the anti-apoptosis nuclear transcription factor B (NF-κB) is not known. Whether escape of the bacteria into the cytosol is sufficient and/or essential for activation of NF-κB is not known. Our results show that F. tularensis subsp. novicida induces sustained nuclear translocation of NF-κB at early time points after infection of human monocytes derived macrophages (hMDMs). The sustained nuclear translocation of NF-κB is defective in the iglC mutant that fails to escape into the cytosol of macrophages. Nuclear translocation of NF-κB by the wild type strain is abolished upon treatment with the NF-κB inhibitor caffein acid phenyl ester. While the wild type strain triggers caspase-3 and caspase-1 activation by 6 h post-infection the iglC mutant is defective in triggering both caspases. In hMDMs treated with the apoptosis-inducing agent, staurosporin, there is an induction of cell death in the iglC mutant-infected macrophages despite reduced frequency of caspase-1 and caspase-3 activity. The wt-infected macrophages are resistant to cell death-induced agent. We conclude that although caspase-1 and capsase-3 are triggered within F. tularensis-infected hMDMs during early stages of infection, cell death is delayed, which is correlated with simultaneous activation of NF-κB. [Copyright &y& Elsevier]

Abstract: Tularemia is a potentially fatal multi-systemic disease of humans and other animals caused by the bacterial pathogen Francisella tularensis. The disease can be transmitted by ticks, biting flies, water exposure, food, and aerosols and occurs around the northern hemisphere including North America, Europe, and Asia. There are several defined species and subspecies, including F. tularensis subsp. tularensis (Jellison Type A) which is pathogenic for rabbits and occurs in North America, F. tularensis subsp. holarctica (Type B) and mediaasiatica which are less pathogenic for rabbits, and F. tularensis subsp. novicida which has been regarded sometimes as the separate species F. novicida. Because it can have a high aerosol-related infection rate, low infectious dose, and ability to induce fatal disease, F. tularensis is considered a potential agent of biological warfare and is classified by the US Department of Health and Human Services as a List A select agent. We discuss microbiological, clinicopathological, epidemiological, and ecological aspects of tularemia. [Copyright &y& Elsevier]

Abstract: A common vole (Microtus arvalis) population peak in Northern Spain in 2007 was treated with large scale application of chlorophacinone, an anticoagulant rodenticide of the indandione family. Voles found dead and trapped alive were collected in treated and untreated areas. Residues of chlorophacinone were analyzed in liver of voles by HPLC–UV. Also, the presence of the pathogen Francisella tularensis was analyzed by PCR in samples of vole spleen. Chlorophacinone (82–3800ng/g; wet weight liver) was only detected in voles found dead in treated areas (55.5%). The prevalence of F. tularensis in voles found dead in treated areas was also particularly high (66.7%). Moreover, chlorophacinone levels were lower in voles that were PCR-positive for F. tularensis (geometric mean [95% CI], 418 [143–1219] ng/g) than in those that were PCR-negative (1084 [581–2121] ng/g). Interactions between pathogens and rodenticides might be considered to reduce the doses used in baits or to avoid the use of the more toxic 2nd generation anticoagulant rodenticides. [Copyright &y& Elsevier]

Abstract: There is very limited evidence concerning the phenotype, function, and homing characteristics of memory T (TM) cells elicited by vaccination against intracellular bacteria in humans. Here we studied TM subsets elicited by exposure to Francisella tularensis in humans as a model of immunity to intracellular bacteria. To this end, TM cells were evaluated in two groups: (1) subjects immunized with live attenuated tularemia vaccine by skin scarification and (2) tularemia naturally infected subjects. In both groups the immune responses were mediated by CD4+ and CD8+ effector TM cells, mostly CD45RA−CD62L− and CD45RA+CD62L−. Based on the expression of CD27, integrins α4/β7, and α4/β1, it is likely that some of these TM cells have lytic potential and the ability to enter both mucosal and non-mucosal sites. Thus, regardless of whether by immunization or natural exposure, tularemia antigens elicited a broad spectrum of specific TM subsets with diverse homing characteristics. [Copyright &y& Elsevier]

Abstract: Francisella tularensis, an intracellular Gram-negative bacterium, is the causative agent of tularemia and a potential bioweapon. Currently, there is no licensed vaccine against this organism. We have characterized the efficacy of a defined F. tularensis subsp. novicida mutant (ΔiglB) as a live attenuated vaccine against pneumonic tularemia. Replication of the iglB mutant (KKF235) in murine macrophages was significantly lower than the wild type novicida strain U112, and exhibited an LD50 greater than 106-fold (>107 CFU vs <10CFU) in an intranasal challenge model. Mice immunized with KKF235 intranasally or orally induced robust antigen-specific splenic IFN-γ recall responses, as well as the production of systemic and mucosal antibodies. Intranasal vaccination with KKF235 protected mice from subsequent homotypic challenge with U112 as well as heterotypic challenge with F. tularensis subsp. holarctica (LVS). Moreover, protected animals also exhibited minimal pathological changes compared with mock-vaccinated and challenged animals. The protection conferred by KKF235 vaccination was shown to be highly dependent on endogenous IFN-γ production. Most significantly, oral immunization with KKF235 protected mice from a highly lethal subsp. tularensis (SCHU S4) pulmonary challenge. Collectively, these results further suggest the feasibility of using defined pathogenicity island mutants as live vaccine candidates against pneumonic tularemia. [Copyright &y& Elsevier]

Abstract: Francisella tularensis, the etiological agent of tularemia, is one of the most infectious bacterial pathogens known. No vaccine is currently approved for public use. Previously, we identified epitopes recognized specifically by T cells obtained from individuals following infection with F. tularensis. Here, we report that a subunit vaccine constructed based upon these epitopes elicited protective immunity in “humanized” HLA class II (DRB1*0401) transgenic mice. Vaccinated mice challenged intratracheally with a lethal dose of F. tularensis (Live Vaccine Strain) exhibited a rapid increase in pro-inflammatory cytokine production and diminished number of organisms in the lungs, and a concurrent increased rate of survival. These results demonstrate the efficacy of an epitope-based tularemia vaccine and suggest that such an approach might be widely applicable to the development of vaccines specific for intracellular bacterial pathogens. [Copyright &y& Elsevier]