Tularaemia.

Tularaemia is a zoonotic bacterial disease of the Northern hemisphere. The causative agent, Francisella tularensis, is spread to humans by direct contact with infected rodents or lagomorphs, aerogenic exposure, ingestion of contaminated food or water, or by arthropod bites. The prevalence of tularaemia shows a wide geographic variation. In some endemic regions, outbreaks occur frequently, whereas nearby rural parts of a country may be completely free. F. tularensis is a facultative intracellular pathogen and its primary mammalian target cell is the mononuclear phagocyte. When tularaemia is acquired via the skin, a primary ulcer is often detected and in general, regional lymph nodes become prominently enlarged. When contracted by inhalation, the disease may present with pneumonia. Nearly as frequent, however, is the development of fever and general illness with no respiratory symptoms and no pulmonary radiological changes. When present, the changes vary widely and may sometimes include hilar enlargement indistinguishable from that of lymphoma. Within an outbreak, the first case of tularaemia is not always readily diagnosed. A decade may have lapsed since the disease was encountered and its existence may be more or less forgotten. The difficulty refers especially to the respiratory form, in which symptoms are less specific. In cases of atypical pneumonia or acute febrile disease with no local symptoms, a history of exposure to hares or rodents or merely living in an endemic region should be sufficient to include tularaemia among differential diagnoses. The microbiological diagnosis of tularaemia relies mainly on serology, and the treatment on broad-spectrum antibiotics. For decades, a live vaccine has been successfully used in risk groups but is presently not available due to difficulties in standardisation.

V gamma 9V delta 2 T cells in human legionellosis.

In humans, expansion of circulating Vgamma9Vdelta2 T cells seems to be a pathophysiological denominator shared by protozoan and intracellular bacterial diseases. The assumption was tested here on legionellosis, a condition conforming to the category but not yet described with respect to gammadelta T cells. Levels of Vgamma9Vdelta2 T cells in peripheral blood were measured at various intervals in 14 subjects undergoing a Pontiac fever-like disease, shown by serological investigation to be caused by Legionella micdadei. In samples obtained 4 to 6 days after the onset of the disease, the mean percentage (+/- the standard deviation) of Vgamma9Vdelta2+ T cells among CD3+ cells was 1.0% +/- 0.5%, compared to 5.0% +/- 3.9% in healthy control subjects (P < 0.001). Thereafter, a pronounced increase occurred and at 2 to 7 weeks after onset, mean peak levels were as high as approximately equal to 15%. During the next 6 months, values slowly declined, although without reaching the normal range. Percentages of gammadelta+ T cells expressing tumor necrosis factor alpha or gamma interferon in response to phorbol myristate acetate were assayed in vitro. At 14 to 16 days after the onset of disease, the expression of both cytokines was increased (P < 0.01), whereas at 5 to 7 weeks, the expression of tumor necrosis factor alpha was decreased (P < 0.05), possibly reflecting modulation of an inflammatory response. In conclusion, Pontiac fever was found to be associated with a pronounced and long-lasting expansion of Vgamma9Vdelta2 T cells, implying that the subset may also be pathophysiologically important in a mild and transient form of intracellular bacterial diseases. Surprisingly, the expansion was preceded by a depletion of circulatory Vgamma9Vdelta2 T cells. Possibly, Vgamma9Vdelta2 T cells are initially recruited to a site of infection before they expand in response to antigen and occur in high numbers in blood.

Long-lasting recall response of CD4+ and CD8+ alphabeta T cells, but not gammadelta T cells, to heat shock proteins of francisella tularensis.

Decades after recovery from tularemia, circulating alphabeta T cells are known to still recognize a variety of membrane proteins of Francisella tularensis. We studied the T cell response to 3 cytoplasmic heat shock proteins of the organism: DnaK, chaperone-60 (Cpn-60) and Cpn-10. Determination of subpopulations of responding T cells was of special interest as it has been suggested that homologs of these conserved proteins may be recognized by human gammadelta T cells. Compared with reference subjects with no history of tularemia or tularemia vaccination, subjects who had been infected with tularemia 10-30 y earlier showed a significantly (p = 0.01) higher proliferative T cell response to all 3 heat shock proteins. In general, the magnitude of responses of CD4 T cells was higher than that of CD8 T cells. By flow cytometry, blast cells were shown to express the alphabeta T cell receptor. Under conditions that allowed vigorous expansion of gammadelta T cells in response to a phosphorylated non-peptide antigen, no expansion of gammadelta T cells occurred in response to DnaK or Cpn60 of F. tularensis. In conclusion, a long-lasting recall response to heat shock proteins of F. tularensis was demonstrated in alphabeta T cells but not in gammadelta T cells. The results support the assumption that human alphabeta T cells recognize bacterial proteins irrespective of the nature or localization of the proteins in the bacterial cell and thereby contribute to the maintenance of a long-lasting broad T cell response based on a wide variety of specificities.

The proportion of circulating gammadelta T cells increases after the first week of onset of tularaemia and remains elevated for more than a year.

In various human intracellular bacterial diseases, an increase of the proportion of circulating Vgamma9Vdelta2 T cells has been observed. The prevalence of the finding among infected subjects and the time course of the elevation remain to be investigated. In the present study, comprising blood samples from a large number of cases of ulceroglandular tularaemia, the percentage of Vgamma9Vdelta2 T cells within the first week of onset of disease (5.3 +/- 0.7% (mean +/- s.e.m.)) did not differ from that of control subjects (5.3 +/- 0. 8%). Thereafter, percentages increased rapidly and within the interval of 8-40 days mean levels were > 20% (P < 0.001). Of 45 individuals sampled within 3 months of onset, 42 showed a percentage of Vgamma9Vdelta2 T cells of > 10%. Significantly increased levels were still recorded at 18 months (13.8 +/- 2.4%; P < 0.05) but not at 24 months (10.2 +/- 2.1%; P > 0.10). Thus, a consistent increase of circulating Vgamma9Vdelta2 T cells was demonstrated in tularaemia. The initial delay and the prolonged course of elevation may suggest a role in immunoregulation and/or immunological memory. Furthermore, the percentage of gammadelta T cells expressing tumour necrosis factor-alpha in response to phorbol myristate acetate was decreased during the first week and up to 40 days after onset, possibly reflecting the modulation of an inflammatory response.

Hospitalization due to Pasteurella multocida-infected animal bite wounds: correlation with inadequate primary antibiotic medication.

Over a 10-y period, patients hospitalized with Pasteurella-induced cat or dog bite-associated wound infection were analysed retrospectively with regard to preceding antibiotic medication. In 10/14 cases, hospitalization was necessitated in spite of prophylactic or therapeutic administration of oral antibiotics. In 1 case, phenoxymethylpenicillin and flucloxacillin had been prescribed. The other patients received flucloxacillin (7 patients), erythromycin, or cefadroxil (1 patient each), agents that are not consistently active against Pasteurella. In conclusion, hospitalization due to Pasteurella-induced animal bite-associated wound infection seemed to be related to the prescription of suboptimal oral antibiotic therapy at a preceding medical consultation.

Ciprofloxacin for treatment of tularemia in children.

BACKGROUND: Children with tularemia are, irrespective of severity of disease, usually subjected to parenteral treatment with aminoglycosides. Based on available susceptibility testing, quinolones might be effective oral alternatives of parenteral therapy. These drugs cause arthropathy in immature animals, but this risk is currently regarded to be low in humans. PATIENTS AND METHODS: In 12 patients (median age, 4 years; range, 1 to 10) with ulceroglandular tularemia, a 10- to 14-day course of oral ciprofloxacin, 15 to 20 mg/kg daily in 2 divided doses, was prescribed. Microbiologic investigations included identification of the infectious agent by PCR and culture of wound specimens, as well as determination of antibiotic susceptibility of isolates of Francisella tularensis. RESULTS: Defervescence occurred within 4 days of institution of oral ciprofloxacin in all patients. After a median period of 4.5 days (range, 2 to 24), the patients were capable of outdoor activities. In 2 cases, treatment was withdrawn after 3 and 7 days because of rash. In both cases a second episode of fever occurred. All children recovered without complications. In 7 cases F. tularensis was successfully cultured from ulcer specimens and tested for susceptibility to ciprofloxacin. MIC values for all isolates were 0.03 mg/l. CONCLUSION: In our sample of 12 patients ciprofloxacin was satisfactory for outpatient treatment of tularemia in children.

Serologic evidence of Puumala virus infection in wild moose in northern Sweden.

Puumala (PUU) virus is the causative agent of nephropathia epidemica, the Scandinavian form of hemorrhagic fever with renal syndrome. The infection is acquired by airborne transmission of PUU virus from its rodent reservoir, the bank vole. Besides serologic data indicating that the virus may spread also to heterologous rodents, there is little information on the susceptibility of wild living animals to PUU virus. We studied the occurrence of antibodies to PUU virus in serum samples from 427 wild-living moose, of which 260 originated from the PUU virus-endemic northern and central parts of Sweden and 167 originated from the southern, nonendemic part of Sweden. Samples from 5 animals showed reactivity in an ELISA for recombinant PUU virus nucleocapsid protein, an immunofluorescent assay, and a neutralization test. These 5 animals all originated from the PUU virus-endemic northern part of Sweden. In conclusion, 5 of 260 moose from the endemic region showed convincing serologic evidence of past PUU virus infection. The seroprevalence was low, suggesting that the moose is subjected to endstage infection rather than being part of an enzootic transmission cycle.

Comparative analysis of PCR versus culture for diagnosis of ulceroglandular tularemia.

PCR and culture were comparatively evaluated for their abilities to demonstrate Francisella tularensis in wound specimens from tularemia patients during an outbreak in Sweden in 1998. For transport of the specimens used for PCR, a buffer solution containing a nuclease inhibitor was used, and for transport of the specimens used for culture, a commercial transport system was selected after experimental comparison of various systems. Of 40 patients with culture- and/or serology-verified ulceroglandular tularemia, PCR detected F. tularensis DNA in 30 (75%) patients, whereas culture detected bacterial growth in 25 (62%) patients. Compared to data from a previous study, the present inclusion of a nuclease inhibitor in the transport medium did not improve the sensitivity of the PCR, whereas the sensitivity of the culture procedure was significantly increased by selection of the system used for transport. Among eight patients with clinically suspected tularemia but with negative serology and culture, specimens from four patients showed detectable DNA. In three of these patients the diagnosis was verified by the demonstration of an F. tularensis-specific T-cell response in vitro. In conclusion, PCR was more sensitive than culture for demonstration of F. tularensis in wound specimens. Besides, we showed that tularemia may proceed without development of serum antibodies, and in these patients, PCR may be of special importance for verification of the diagnosis.

Booster vaccination with recombinant hepatitis B vaccine four years after priming with one single dose.

We here studied the antibody response to a booster dose four years after the administration of one single dose of recombinant HB vaccine. Before receiving the booster dose, levels of protective antibodies (anti-HBs) were generally low and 24/41 (59%) individuals lacked detectable antibodies (< 1 IU/L). Within 14 d of booster vaccination, 36/38 (95%) vaccinees showed levels of antibodies > 100 IU/L. Notably, these levels were at least as high as those of a reference group 12 months after initiation of vaccination according to the standard three-dose vaccination at intervals of 0, 1 and 6 months. In conclusion, one single dose of HB vaccine seemed to confer on young healthy individuals a well preserved B cell memory, disclosed as a rapid and strong antibody response to a second dose four years later.

A minority of seropositive wild bank voles (Clethrionomys glareolus) show evidence of current Puumala virus infection.

Bank voles (Clethrionomys glareolus) serve as the reservoir for Puumala (PUU) virus, the aetiologic agent of nephropathia epidemica. The animals are believed to be persistently infected and the occurrence of serum antibodies is usually taken as an evidence of active infection. We found serum antibodies to PUU virus in 42 of 299 wild bank voles captured in a PUU virus endemic area. PUU virus RNA was demonstrated in lung specimens of 11 of these 42 animals and in 2 of them antigen was also found. Thus in the lungs of 31 of 42 seropositive animals neither PUU virus RNA nor antigen was detected. In 2 of 257 seronegative animals, lung specimens showed presence of PUU virus antigen and RNA. Isolation of PUU virus from lung tissue was successful in all 4 antigen-positive bank voles but in none of 16 tested antigen-negative animals. In conclusion, only a minority of bank voles with serum antibodies to PUU virus showed evidence of current infection.

Prevalence of antibodies specific to Puumala virus among farmers in Sweden.

OBJECTIVES: The Puumala virus is the causative agent of nephropathia epidemica, a European form of hemorrhagic fever with a renal syndrome. From its reservoir in bank voles, the virus is spread by airborne transmission to humans. Occupational risks for the acquisition of nephropathia epidemica are not well defined. The prevalence of serum antibodies to Puumala virus was determined for Swedish farmers. From a comparison of the prevalence among farmers from various parts of the country, the assumption that Puumala virus occurs endemically only in the northern and central parts of Sweden was also tested. METHODS: Serum samples from 910 farmers and 663 referents living in various rural parts of Sweden were tested with an enzyme-linked immunosorbent assay, using a recombinant nucleocapsid protein of Puumala virus as the antigen. RESULTS: North of a latitude of 59 degrees N, the prevalence of Puumala virus antibodies was significantly higher among farmers (12.9%) than among referents (6.8%). In the southern areas, antibodies to Puumala virus were rare, and altogether only 2 of 459 persons had antibodies. Seropositive persons did not differ from seronegative ones with regard to blood pressure, and they did not comprise cases of chronic renal disease. CONCLUSIONS: Serological evidence confirmed that the exposure of humans to Puumala virus is firmly restricted to the northern and central parts of Sweden. In addition the evidence indicated that, in this region, farming is associated with an increased risk of contracting hantavirus infection.

Development of humoral cross-reactivity to the nucleocapsid protein of heterologous hantaviruses in nephropathia epidemica.

A hantavirus infection is followed by a prominent antibody response to the viral nucleocapsid protein. Antibodies from patients infected with one hantavirus cross-react to varying degrees with the nucleocapsid protein of other viruses of the genus. We studied the cross-reactivity in serially obtained blood samples from 17 patients with nephropathia epidemica, a European form of hemorrhagic fever with renal syndrome caused by Puumala virus. Recombinant truncated nucleocapsid protein (aa 1-117) of Puumala virus and four other hantaviruses, Hantaan, Seoul, Dobrava and Sin Nombre viruses, were used as antigens in an indirect ELISA. In most patients, an IgG response to the Puumala virus derived recombinant protein was detected within 2-8 days of onset of disease, remained high for 2-5 months, and declined gradually within 2-3 years. All patients had IgG antibodies cross-reacting with the nucleocapsid protein of Sin Nombre virus. The ratio of the ELISA values obtained with Sin Nombre vs. Puumala virus protein as antigen increased with time after onset of disease. To a lesser extent, cross-reacting IgG antibodies also occurred to Hantaan, Seoul, and Dobrava virus antigens. In the acute phase of the disease, two patients showed IgG antibodies to one or more of these viruses whereas 2-5 months later, 11 of 16 patients had IgG antibodies to all three viruses. IgM and IgA responses to the nucleocapsid protein of Puumala virus were transitory and cross-reactivities were weak. In conclusion, after onset of nephropathia epidemica the IgG response to the Puumala virus nucleocapsid protein was associated with a gradually increasing cross-reactivity to the nucleocapsid protein of heterologous hantavirus. Our findings have implications for the interpretation of serological data, both in the diagnostics of nephropathia epidemica and in seroprevalence studies.

Impaired pulmonary function in patients with hemorrhagic fever with renal syndrome.

Pulmonary and cardiac functions were investigated in 13 patients hospitalized with nephropathia epidemica, a European form of hemorrhagic fever with renal syndrome. As compared with reference values, the patients' diffusion capacity for carbon monoxide was decreased (P = .002) and pulmonary clearance of inhaled technetium-99m-labeled diethylenetriamine pentaacetic acid was increased (P = .002). In four of 11 patients, arterial blood gas analysis disclosed a reduction in partial pressure of O2 (< 10 kPa) and oxygen saturation (< 94%). In three of 13 patients, chest radiography revealed interstitial infiltrates or pleural effusions. Lung volumes and expiratory flow rates of the patients were not significantly changed. By electrocardiography and echocardiography, no significant cardiac dysfunction was demonstrable. The pulmonary dysfunction was best explained by an alveolocapillary lesion. The two hantavirus-caused clinical syndromes, hemorrhagic fever with renal syndrome and hantavirus pulmonary syndrome, may be pathophysiologically more similar than appears from the clinical presentations.

Increased plasma levels of soluble CD23 in haemorrhagic fever with renal syndrome; relation to virus-specific IgE.

In 15 consecutive patients hospitalized with nephropathia epidemica, a European form of haemorrhagic fever with renal syndrome (HFRS) caused by Puumala virus, plasma concentrations of soluble CD23 (sCD23) and Puumala virus-specific IgE were determined. In the acute phase of illness, 11/15 patients had increased sCD23 levels (> 91 U/ml), whereas in convalescence, values of 8/10 patients were normalized. Maximal sCD23 values were correlated to maximal concentrations of Puumala virus-specific serum IgE (r = 0.597; P = 0.025). The results are compatible with a known ability of sCD23 to augment IgE production.

Identification of proteins of Francisella tularensis induced during growth in macrophages and cloning of the gene encoding a prominently induced 23-kilodalton protein.

The adaptation of facultative intracellular bacteria to host macrophages involves regulation of the synthesis of bacterial proteins. We analyzed the protein synthesis of Francisella tularensis LVS growing intracellularly in the macrophage-like murine cell line J774 and extracellularly in culture medium. After pulse-labeling with [35S] methionine and separation by one- and two-dimensional polyacrylamide gel electrophoresis, induction of a few proteins during intracellular growth was demonstrated. One of them, a 23-kDa protein, was prominently induced in the macrophages and also when extracellularly growing F. tularensis was exposed to hydrogen peroxide. After isolation of the 23-kDa protein from a preparative two-dimensional gel, a 22-amino-acid N-terminal peptide and two peptides obtained by trypsin digestion were sequenced. Based on the sequences, degenerate oligonucleotides were constructed for use as primers in a PCR. Hybridization of amplified DNA to XbaI-digested LVS DNA identified the gene of the 23-kDa protein in a 1.3-kb DNA fragment. Nucleotide sequence analysis revealed an open reading frame encoding a putative protein of a calculated molecular mass of 22.2 kDa. The open reading frame was preceded by a sequence typical of ribosome-binding sites in Escherichia coli. The amplified gene was successfully expressed by the pTrc99A vector in E. coli under control of the trc promoter. The gene product showed the same mobility and immunoreactivity as the 23-kDa protein of F. tularensis. The deduced amino acid sequence showed no significant homology with protein sequences in current data banks. Thus, intracellular growth of F. tularensis in macrophages was associated with prominent upregulation of a novel 23-kDa protein.