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Disease tolerance can preserve host homeostasis and limit the negative impact of infections. We report that vaccinated mice survived pulmonary challenge with the extremely virulent SchuS4 strain of Francisella tularensis for at least 100 days, despite the persistence of large numbers (~10(4)) of organisms. Transfer of 100 of these resident bacteria to naive animals caused 100% lethality, demonstrating that virulence was maintained. Tissue damage in the lung was limited over the course of infection and was associated with increased levels of amphiregulin. Mice depleted of CD4(+) cells had reduced amphiregulin and succumbed to infection. In addition, neutralization of interferon-γ or depletion of CD8(+) cells resulted in increased pathogen loads, bacteremia, and death of the host. Conversely, depletion of Ly6G(+) neutrophils had no effect on survival and actually resulted in reduced bacterial levels. Understanding the interplay between host resistance and disease tolerance will provide new insights into the understanding of chronic infectious diseases.

Francisella tularensis is a gram-negative coccobacillus that causes tularemia, a highly infectious zoonotic disease. Because of its high infectivity, ease of dissemination, and ability to cause severe disease and death, F. tularensis is designated by the Centers for Disease Control and Prevention as a category A select bacterial agent [1]. Differentiation of the 4 closely related subspecies of F. tularensis (tularensis, holarctica, mediaasiatica, and novicida) depends on epidemiology, infection source, and clinical manifestations [2]. F. tularensis subsp tularensis (type A) is highly virulent to all mammalian species, including humans, with mortality rates of 30%–60% in systemic infection following inhalation. F. tularensis subsp holarctica (type B) is less virulent to humans but can cause chronic debilitating illness. Both type A and type B are highly virulent in mice. The F. tularensis live attenuated vaccine strain (Ft.LVS), which was derived by serial in vitro passage of a type B strain [3], has been used to immunize high-risk groups, but currently is not licensed as a vaccine because of an incomplete understanding of its attenuated virulence (at a genetic level), protective properties, and untoward reactions. Multiple strategies have been employed to develop a protective vaccine against tularemia, including the use of killed whole bacteria [4], subunits [5–9] and live attenuated strains [10–12]. However, inadequate knowledge of the protective antigens and virulence mechanisms of F. tularensis [13–15] still renders it difficult to develop a fully satisfactory vaccine. The lipopolysaccharide (LPS) of F. tularensis has attracted considerable interest because of its unusual biological and structural properties and its key role in virulence. Unlike the LPSs of other gram-negative bacteria, that of F. tularensis does not induce innate immune responses [16, 17]. However, the O-polysaccharide (O-PS) portion of the LPS, when used in a glycoconjugate vaccine, reportedly plays a major role in immunity [18, 19] by inducing specific protective antibodies [20]. Cooperation of the humoral and cellular arms of the immune system is essential for effective protection against tularemia [21, 22]. We recently used the avirulent, O-PS-negative strain (Ft.LVS::δwbtA) deleted in wbtA (accession no. {"type":"entrez-nucleotide","attrs":{"text":"DQ353940","term_id":"90654210","term_text":"DQ353940"}}DQ353940) together with a glycoconjugate (tetanus toxoid–O-PS) as a combinatorial vaccine [20] in which the glycoconjugate-induced humoral immunity and the wbtA mutant accounted for cellular immunity. This combination of immunogens conveyed enhanced protection against both type A (SchuS4) and type B (FSC108) strains (intradermal infection) as well as partial protection (enhanced over that provided by either component alone) against intranasal infection with type A strains. The wzy gene (FTL_0598) of F. tularensis is located on the genome at the O-antigen locus. This gene encodes an O-antigen polymerase (Wzy) that is genetically and structurally related to the wzy genes responsible for the polymerization of heteropolymeric O antigens in gram-negative bacteria [23, 24]. We have functionally characterized a wzy deletion mutant (Ft.LVS::Δwzy) and have shown that Wzy is responsible for polymerization of the O-PS during LPS synthesis [24]. An important point is that the mutant expresses only 1 repeating unit of O antigen on its cell surface [24], whereas the parental Ft.LVS expresses multiple repeating units. Herein we show, in a mouse model of infection, that the Ft.LVS::Δwzy is a good candidate vaccine against tularemia. Ft.LVS::Δwzy is attenuated by at least 7 log10 compared with the parental Ft.LVS. Moreover, compared with the Ft.LVS::ΔwbtA, the Ft.LVS::Δwzy is significantly more protective against type A strains. While the Ft.LVS::ΔwbtA induced only cellular immunity [19, 20], the Ft.LVS::Δwzy mutant induced both cellular and humoral immunity, as its nonrepeating single unit of O antigen induced protective antibodies reacting with full-length O-PS. The Ft.LVS::Δwzy mutant offers some significant advantages over the combinatorial vaccine (ie, the O-PS glycoconjugate plus the Ft.LVS::ΔwbtA), as it is significantly more protective against type A strains, and is much easier to administer.

(See the editorial commentary by Conlan, on pages 6–8.) Francisella tularensis is the causative agent of tularemia and is classified as a category A biodefense agent by the Centers for Disease Control and Prevention because of its highly infectious nature. F. tularensis infects leukocytes and exhibits an extracellular phase in the blood of the host. It is unknown, however, whether F. tularensis can infect erythrocytes; thus, we examined this possibility in vivo and in vitro. In the murine model of pulmonary type A tularemia, we showed the presence of intraerythrocytic bacteria by double-immunofluorescence microscopy and ex vivo gentamicin protection of the purified erythrocyte fraction. In vitro, F. tularensis invaded human erythrocytes, as shown in the gentamicin protection assays, double-immunofluorescence microscopy, flow cytometry, scanning electron microscopy, and transmission electron microscopy with immunogold labeling of the bacteria. Additional in vitro tests indicated that serum complement-dependent and complement-independent mechanisms contribute to erythrocyte invasion. Our results reveal a novel intraerythrocytic phase during F. tularensis infection.

The ability of Francisella tularensis to replicate in macrophages has led many investigators to assume that it resides primarily intracellularly in the blood of mammalian hosts. We have found this supposition to be untrue. In almost all cases, the majority of F. tularensis recovered from the blood of infected mice was in plasma rather than leukocytes. This distribution was observed irrespective of size of inoculum, route of inoculation, time after inoculation, or virulence of the infecting strain. Our findings yield new insight into the pathogenesis of tularemia and may have important ramifications in the search for anti-Francisella therapies.

Background. We aimed to evaluate the potential association of mosquito prevalence in a boreal forest area with transmission of the bacterial disease tularemia to humans, and model the annual variation of disease using local weather data. Methods. A prediction model for mosquito abundance was built using weather and mosquito catch data. Then a negative binomial regression model based on the predicted mosquito abundance and local weather data was built to predict annual numbers of humans contracting tularemia in Dalarna County, Sweden. Results. Three hundred seventy humans were diagnosed with tularemia between 1981 and 2007, 94% of them during 7 summer outbreaks. Disease transmission was concentrated along rivers in the area. The predicted mosquito abundance was correlated (0.41, P , .05) with the annual number of human cases. The predicted mosquito peaks consistently preceded the median onset time of human tularemia (temporal correlation, 0.76; P , .05). Our final predictive model included 5 environmental variables and identified 6 of the 7 outbreaks. Conclusions. This work suggests that a high prevalence of mosquitoes in late summer is a prerequisite for outbreaks of tularemia in a tularemia-endemic boreal forest area of Sweden and that environmental variables can be used as risk indicators.

The humoral and cell-mediated immune responses to the facultative intracellular pathogen Francisella tularensis were studied in subjects who had tularemia 25 years earlier. Serum agglutinin titers were low. Of 53 subjects, only 2 had a titer > 40. The F. tularensis-induced T cell response, on the other hand, was vigorous and in fact similar in magnitude to the response to purified protein derivative (PPD) of Mycobacterium tuberculosis. T cells from 44 of 52 subjects showed a significant (P < .05) proliferative response in vitro to each of four membrane proteins of F. tularensis. Only 2 subjects did not respond to any of the proteins. In most cultures from 18 subjects tested, interferon-gamma was produced in response to the proteins. During the last 25 years, tularemia has been reported only rarely in the region where the subjects lived, suggesting that an antigen-specific T cell response may persist after tularemia independently of reexposure to F. tularensis.

A whole-blood lymphocyte stimulation test was used in the diagnosis of 200 cases of tularemia. Ninety-one of the patients involved had ulceroglandular infection, 81 had pulmonary infection, 13 had glandular infection, four had oropharyngeal infection, and one had oculoglandular infection. The clinical picture was obscure in the remaining 10 cases. Results of the lymphocyte stimulation test became positive in 21.3% of cases during the first week, when an agglutinating titer of greater than or equal to 1:80 was found in 2.1% of patients. During the second week, these proportions were 96.8% and 53.3%, respectively. In only one patient with tularemia (0.5%) did the stimulation test result remain negative, and only one (0.5%) of the 211 control patients had a positive result in the first test. The development of cell-mediated immunity followed the same pattern in patients with ulceroglandular tularemia as in those with pulmonary tularemia.

To investigate the effects of strenuous forced exercise on the course and complications of a bacterial infection and on myocardial responses and performance capacity, rats with tularemia (characterized by pyogranulomatous hepatic and splenic lesions) were exercised by swimming on days 0-6 of infection. Levels of glutamic oxaloacetic and pyruvic transaminases in plasma, densities of pyogranulomatous lesions, and bacterial counts in blood, liver, and spleen were similar in exercising and resting rats. Although a few exercising rats showed an unusual dissemination of infection, the antibody responses were similar in rest and exercise. Plasma concentrations of beta-glucuronidase, lysozyme, and alpha sub 2 -macrofetoprotein were higher with exercise, a result that indicated that more vigorous stress responses were elicited with exercise than with infection alone. Physical performance capacity was reduced by the infection, but forced daily exercise limited this reduction substantially and counteracted the myocardial protein-degrading effects of infection. Thus, exercise evoked normal training responses even during this generalized infection.

Between May 9 and July 3, 1979, 12 cases of glandular or ulceroglandular tularemia occurred in residents of the Crow Indian Reservation in southcentral Montana; only 13 cases had been reported from this geographic area in the preceding 25 years. The illness was mild, characterized by fever and cervical or occipital adenopathy. Systemic symptoms were self-limited although residual lymphadenopathy was common. Francisella tularensis was isolated from ticks (Dermacentor variabilis), the suspected vector. The strains of F tularensis did not ferment glycerol and thus were identified as type B rather than the more virulent type A. None of 83 adults hospitalized in an urban area 50 miles from the reservation had agglutination titers of antibody to F tularensis of greater than or equal to 1:40 compared with eight of 77 patients at the reservation hospital (P less than 0.01). Mild tularemia in reservation residents may have gone unrecognized; similar illness due to type B F tularensis may occur elsewhere.

birds which are known to be naturally infected with Past. tularensis (Burroughs et al, 1945) serve as hosts of argasid ticks, especially of the genus Ornithodoros. Kamil and Bilal (1938) reported transmission of Past. tularensis by experimentally infected Ornithodoros lahorensis. Davis (1940), in studies of ticks of the genus Ornithodoros as potential vectors of various infectious agents, found that Past. tularensis may persist in the tissues of 0. turicata and 0.

Health Nurses, including Mrs. Thomas Haley, Mrs. Elaine Clement, and the Misses Mildred Walter, Lovina Woodrow, Elizabeth Van Creveld and Inez Monaghan, whose itinerant nursing activities in remote Alaska villages made this study possible. The contributions of the Alaskan Division of Health, particularly those of Mr. Ralph B. Williams and Mr. Frank P. Pauls, are appreciated. The authors would also like to thank Dr. Karl R. Reinhard for help in obtaining blood samples, Mr. Raymond Thrailkill for his technical assistance, and Mrs. Phillip Judd for her statistical clerical help. * U. S. Public Health Service, National Institute of Allergy and Infectious Diseases, Rocky Mountain Laboratory, Hamilton, Montana. t U. S. Public Health Service, Communicable Disease Center, Tuberculosis Program, Washington 25, D. C. mia in a man from Northway in central Alaska who had trapped muskrats shortly prior to illness. Subsequently, at least 3 other cases were identified and

Francis and Felton4 have proposed a measure of virulence, based on the susceptibility of mice, guniea pigs, and rabbits to infection with this organism, which appears to be the most direct and valid of any that have been applied. Approximately 1 to 10 organisms of maximum virulence are sufficient to cause fatal infection in mice, guinea pigs, and rabbits. Diminished virulence first shows itself in the failure of the organisms to kill rabbits. This method of as

hospitalized and nonhospitalized volunteers. After exposure, men continued to work normally during incubation periods; decrements in performance first appeared in coincidence with onset of illness and became maximal when symptoms were greatest. Patterns of decrements were similar in bacterial and viral illnesses, but the degree was greater on the average with tularemia than with sandfly fever (drops to 69% and 80% of baseline, respectively). Measured decrements were general, rather than specific to any of several behavioral functions, and average decrements were clearly related in timing and magnitude to the presence and severity of illness. Estimates of motivation and extensive clinical and psychological tests provided no insight with regard to differences among individual volunteers in the ability to maintain performance at levels observed before illness; such differences were essentially maximal in range, even with as few subjects as the 16 in the studies on tularemia. Among subjects who were equally febrile, some showed no decrements in performance, whereas others were unable to work at all.

Widespread alterations occur in the metabolism of amino acids of the host during an infectious illness, but little is known about the specific changes. Tryptophan, a subject of a variety of studies, is unique in that its metabolites contribute to the diazo reaction [1]. Ehrlich [2] reported strongly positive diazo reactions during typhoid fever, and the test was used widely in the diagnosis of this infection before the era of bacterial identification [3]; in fact, it is still recommended as a useful test in the diagnosis of typhoid fever in underdeveloped regions [4]. Altered metabolism of tryptophan has been observed in other infectious illnesses [5-8]. In studies reported by Berry and Smythe [9], it was shown that tryptophan oxidase (TO), the hepatic enzyme which may limit the rate of tryptophan metabolism via the kynurenine pathway, exhibited reduced activity in mice during experimental infection with Salmonella typhimurium. Depression of TO activity was also reported during overwhelming staphylococcal infection [5]. While similar depression of TO activity occurred late in the course of pneumococcal septicemia in mice, a significant induction of the enzyme was noted during the incubation period of infection [10]. Altered tryptophan metabolism has also been implicated in the pathogenesis of endotoxin shock in experimental animals [11]. It seemed, therefore, appropriate to investigate the relationship between enzymatic degradation of tryptophan and the clinical responses in man during the course of several different infectious illnesses.

tularensis. Skin test surveys (Hopla, 1960; Philip et al, 1962) provided additional evidence suggesting frequent infection of Alaskan natives with F. tularensis. However, clinical tularemia has seldom been recognized in these areas, and a major attempt to identify natural foci of infection in central Alaska was unsuccessful (Hopla, 1960; Downs, 1960). Since an extensive reservoir of tularemia has yet to be identified, the possibility of infection by an unknown, but antigenically related, agent cannot be completely disregarded. The present study was undertaken in Montana where clinical tularemia has

Significant changes in the whole blood amino acids of volunteers with respiratory-acquired Pasteurella tularensis (SCHU-S4) infection have been described. A decrease in whole blood amino acids occurred 12 to 60 hours after exposure and prior to the time that clinical symptomatology was apparent. The factors responsible for this decreased amino acid concentration were discussed but remain undefined. These changes were specifically related to infection, in that sham-exposed and similarly treated subjects did not manifest a similar response. The pattern of response was such that, in retrospect, it appeared to have predictive value for determining which subjects had been infected.