Correspondence: Roland Grunow, Centre for Biological Safety, Robert Koch-Institut, Nordufer 20, D-13353 Berlin, Germany. Tel.: +49030/18 754 2100; fax: +49030/18 754 2110; e-mail: email@example.com
In 2005 and 2006, Francisella tularensis unexpectedly reemerged in western Germany, when several semi-free-living marmosets (Callithrix jacchus) in a research facility died from tularemia and a group of hare hunters became infected. It is believed that hunters may have an elevated risk to be exposed to zoonotic pathogens, including F. tularensis. A previous cross-sectional study of the German population (n=6883) revealed a prevalence of 0.2%. Here, we investigated 286 sera from individuals mainly hunting in districts with emerging tularemia cases (group 1) and 84 sera from a region currently not conspicuous for tularemia (group 2). Methods included standard enzyme-linked immunosorbent assay (ELISA), Western blot analysis and indirect immunofluorescence assay. We found five out of the 286 hunters (1.7%; 95% CI 0.6–4.0%) in group 1 positive with standard ELISA and Western blot, but none in the Berlin area (group 2; 95% CI 0–0.04%). Group 1 showed an elevated risk for hunters to be seropositive for F. tularensis compared with the cross-sectional study (OR=7.7; P<0.001). This indicates a higher prevalence for tularemia in hunters of a suspected endemic region of Germany.
Tularemia is a zoonosis caused by a gram-negative, pleomorphic, nonmotile bacterium Francisella tularensis, a potent intracellular pathogen with a potential use in warfare and bioterrorism. Because of its high infectivity and low infectious dose, F. tularensis has been classified as a Category A select agent (Darling et al., 2002). Francisella tularensis is divided into four subspecies: tularensis, mediasiatica, holarctica and novicida, which differ in their virulence and were found in different regions of the world. In Europe, the subspecies holarctica has been found almost exclusively. With the exception of novicida, the subspecies remain phylogenetically closely related and antigenically similar (Garaizar et al., 2006). It occurs in mammals including humans, whereas the sources of infection are mostly rodents, with arthropods being the primary vector (Ellis et al., 2002). Infection is often acquired by handling animal skins or carcasses, but it is also possible to acquire the disease from drinking contaminated water or by eating uncooked contaminated meat (Higgins et al., 2000). However, in regions where tularemia is endemic, antibodies to F. tularensis are often detected in the sera of wild animals (Morner & Sandstedt, 1983; Frolich et al., 2002), and outbreaks of the disease occur frequently in parallel in wild animals and humans (Ellis et al., 2002).
Tularemia is a very rare zoonosis in Germany. Because the new Infectious Disease Control Act (Infektionsschutzgesetz) came into force in 2001, between three and five cases have been reported to the Robert Koch Institute in Berlin each year (Hofstetter et al., 2006). Interestingly, 12 cases of tularemia have been reported in Germany until now for 2007. Depending on the variable clinical appearance of the disease, it is possible that it is not recognized as such and a high estimated number of unreported cases is assumed. This assumption was supported by a cross-sectional study with more than 6000 sera revealing a seroprevalence of 0.2% in the German population (Porsch-Ozcurumez et al., 2004). Because little is known about the current prevalence of tularemia in Germany among humans and animals, we wanted to know more about its distribution based on the outbreaks that recently occurred. During one outbreak in 2005 in Hesse, nine hunters showed clinical symptoms of tularemia and were serologically confirmed (Hofstetter et al., 2006). Additionally, F. tularensis was identified as a cause of die-off among a colony of semi-free-living common marmosets (Callithrix jacchus) in Lower Saxony. During the outbreak all five sick animals died, whereas 62 were not infected. The outbreak areas in West Germany were suspected for the long-term presence of the tularemia bacterium (Splettstoesser et al., 2007). In a retrospective study of die-offs inside a colony of macaques in Lower Saxony (Goettingen), the natural prevalence of Francisella in West Germany was underlined (Matz-Rensing et al., 2007).
Even though early identification of the pathogen is important, isolation by cultivation, immunologic detection of antigens, or molecular approaches are not always successful or suitable (Junhui et al., 1996; Sjostedt et al., 1997; Grunow et al., 2000). Although antibodies are detectable 1–2 weeks after infection, agglutination assays and immunofluorescence techniques are mostly used as serological tests in the diagnosis of acute tularemia and for epidemiological studies (Schmitt et al., 2005). Enzyme-linked immunosorbent assay (ELISA) as screening and Western blot as confirmational assay have been established in recent years for the identification of tularemia antibodies, mostly on the basis of lipopolysaccharide recognition (Porsch-Ozcurumez et al., 2004; Schmitt et al., 2005). Lipopolysaccharide of F. tularensis is the main target for the development of species-specific antibodies (Aronova & Pavlovich, 2001) and it was shown that these antibodies did not recognize lipopolysaccharide of potentially cross-reacting bacteria (Grunow et al., 2000).
The aim of this study was to investigate the occurrence of tularemia in different parts from Germany. Therefore, we used the standard methods ELISA and Western blot for the detection of F. tularensis antibodies in serum collections derived from hunters from putative endemic and nonendemic regions, respectively. Additionally, confirmed positive sera were analyzed by indirect immunofluorescence assay (IFA) on inactivated bacteria.
Materials and methods
Sample collection and statistics
For validation of the in-house assay, a total of 224 anonymized negative sera were obtained at a routine medical check-up from mainly healthy young German adults as a negative group. The sera were collected as remaining volumes after completing the requested medical laboratory investigation. Additionally, we used clinical sera from patients suspected to have tularemia (n=177) also for validation, kindly provided from Alain Le Coustumier, Cahors, France. The French sera were tested on tularemia antibodies beforehand by micro-agglutination assay. At our laboratory these sera were examined by lipopolysaccharide ELISA and Western blot. A total of 286 hunters' sera (group 1) were collected randomly on a special hunting exhibition in Dortmund (western part of Germany) in February 2006. In April 2006, we enrolled 84 municipal hunters from Berlin (group 2). Demographic information (age and sex), frequencies of contact to hares, main hunting districts and possible clinical symptoms were assessed by a standardized questionnaire. Ninety-five percent confidence intervals (95% CI), P-values and odds ratio (OR) were calculated using spss 14 software (SPSS, Chicago).
The live vaccine strain (LVS) of F. tularensis spp. holarctica (ATCC29684) was grown for 3 days at 37 °C in a 5% CO2, humid atmosphere on heart–cystein–blood agar. After harvesting of colony material into 0.85% NaCl, bacteria were inactivated by the addition of 1% formamide (end concentration) overnight at room temperature. Cells were adjusted to OD600nm=1.0 and OD600nm=2.4 for different applications.
Standard lipopolysaccharide ELISA
Lipopolysaccharide ELISA was essentially performed as described elsewhere (Porsch-Ozcurumez et al., 2004; Schmitt et al., 2005). In brief, flat-bottomed 96-well polystyrene plates (PolySorp, NUNC, Wiesbaden, Germany) were coated by passive absorption with 50 μL per well lipopolysaccharide of F. tularensis (Micromun, Greifswald, Germany) in a concentration of 0.5 μg mL−1 in carbonate/bicarbonate buffer, pH 9.0, for 1 h at 37 °C. After three washes with phosphate-buffered saline (PBS) (pH 7.3) with 0.05% Tween 20 (PBS-T), wells were blocked with 75 μL block and dilution buffer containing 10% goat serum in PBS-T for 90 min at 37 °C. Sera and control sera were diluted 1/500 and 50 μL was added after three washes with PBS-T in duplicate wells for 1 h at 4 °C. Wells were washed five times and 50 μL of polyvalent goat anti–human IgA–IgM–IgG horseradish peroxidase conjugated secondary antibody (Dianova, Hamburg, Germany) was added with 1 μg mL−1 for 1 h at 37 °C. After incubation, wells were washed with 300 μL PBS-T five times. The wells were filled with 200 μL of O-phenylendiamine (OPD; Sigma Aldrich, Taufkirchen, Germany) as substrate; reaction was stopped after 10 min by the addition of 50 μL 2.5 M sulfuric acid. Optical density values were then recorded at 492 nm with a Sunrise Plate Reader (Tecan Instruments, Crailsheim, Germany), interfaced with a computer. Samples were considered positive if they showed an OD greater than the cut-off. The cut-off was calculated from the mean OD of negative control sera plus two SD.
LVS suspension was inactivated with 1% formalin overnight and adjusted to an OD600nm of 2.4. Subsequently, 150 μL was treated with 50 μL of 4 × Laemmli sample buffer [10% sodium dodecylsulfate (SDS) (w/v), 35% glycerol (v/v), 0.5 M Tris–HCl, pH 6.8, 3.5% 2-mercaptoethanol, 5% bromophenol blue] for 10 min at 70 °C. After 15 min of boiling, the suspension was centrifuged for 10 min at 7000 g. The soluble fraction was subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) using a 12% separating gel and a 5% stacking gel with a preparative comb at 20 mA in a minigel chamber (Biometra, Goettingen, Germany) for 1.5 h.
The gel was soaked shortly in blotting buffer (50 mM Tris base, 39 mM Glycine, 0.037% SDS, 20% methanol), and the bacterial antigens were transferred by semi-dry blotting (Phase, Luebeck, Germany) to a polyvinylidene difluoride (PVDF) membrane (ImmobilonP, Millipore, Billerica, MA). Unspecific binding sites on the membrane were blocked with 10% skimmed milk in Tris-buffered saline (pH 7.6) with 0.1% Tween 20 (TBS-T) for 30 min at room temperature. The membrane was incubated in a mini blotting apparatus (Biometra, Goettingen, Germany) according to the manufacturer's instructions with different sera per slot diluted 1/500 in 3% skimmed milk in TBS-T for 1 h at room temperature. After washing with TBS-T via the manifold unit, the slots were filled with a polyvalent goat anti-human IgA–IgM–IgG horseradish peroxidase conjugate (Dianova, Hamburg, Germany) and incubated for 1 h at room temperature. Following another rinse via the manifold, the membrane was developed with enhanced chemoluminescence (ECL; Pierce/Perbio, Bonn, Germany). Sera were considered positive when they showed the typical lipopolysaccharide banding pattern.
For the IFA, 10 μL of a 1/100 dilution of formalin-inactivated F. tularensis spp. holarctica strain LVS with an OD600nm of 1.0 was placed on immunofluorescence slides cleaned with ethanol (BioMerieux, Marcy I'Etoile, France). Slides were dried for 2 h at room temperature and fixed with ice-cold methanol for 15 min. Blocking was done with 5% goat serum (Gibco/Invitrogen, Karlsruhe, Germany) in 1% bovine serum albumin/PBS for 30 min at room temperature. Sera were diluted 1/150 and 25 μL was added and incubated for 30 min at 37 °C. After washing with PBS the Alexa®488 conjugated secondary goat anti-human antibody (Molecular Probes, Karlsruhe, Germany) was added for 30 min at 37 °C and slides were washed with PBS. Mounting was done with Prolong Gold Antifade plus 4′,6-diamidino-2-phenylindole (DAPI) (Molecular Probes) according to the manufacturer's instructions and preparations were examined by fluorescence microscopy.
Data were acquired with an AxioCam MR camera (Zeiss) driven by axiovision software (version 4.4, Zeiss) and processed using Adobe Photoshop CS2 (Adobe Systems) software.
For evaluation purposes we used 177 clinical sera from France for our in-house assay and tested 65 (36.7%) positive in lipopolysaccharide ELISA as a prescreening method (Table 1), which is in accordance with the microagglutination assay (Koskela & Salminen, 1985) used as a screening method by the provider. The level of accordance between Western blot and agglutination assay was 64%, between Western blot and ELISA 78%, whereas the divergence was due to false positive results in the agglutination assay as also seen in ELISA.
Table 1. Detection of antibodies against Francisella tularensis in different serum collections
To confirm the ELISA results, we further examined positive tested sera by Western blot and found 50 (29.4%) showing the typical lipopolysaccharide banding pattern. Sera from mainly healthy people were used as the negative group to validate the serological assays. We found 12 (5.4%) to be positive in ELISA, but confirmed just one (0.4%) in Western blot. From the negative group, we defined the cut-off values for lipopolysaccharide ELISA as OD<0.25 (mean+1 SD) for negative samples, from OD 0.25 to 0.4 were suspicious and sera with ODs≥0.4 (mean +2 SD) were positive. ELISA suspicious and positive sera were admitted to confirmatory Western blot. Sera showing high background or unspecific bands in Western blot but no lipopolysaccharide ladder were stated as negative (not shown).
To investigate the prevalence of tularemia among individuals from risk groups, we tested 286 serum samples from German hunters living mainly in the western regions of Germany (Fig. 1, group 1, green boxes) and 84 serum samples from hunters living in an area surrounding Berlin (group 2, blue boxes) for the presence of antibodies against F. tularensis by standard lipopolysaccharide ELISA. Eighteen sera (6.3%) from hunters group 1 were tested positive in ELISA, whereas five (1.7%; 95% CI 0.6–4.0%) out of 18 could be confirmed by Western blot, showing the typical lipopolysaccharide ladder on F. tularensis extract (Fig. 2) with mean intensities of 2 or 3. Additionally, these sera were verified by IFA. Two hunters, namely 61 and 114, showed weak Western blot signals with mean intensities of 1 and also weak IFA signals and were stated as borderline, but not calculated as positive. The sera of hunters 124 and 206 showed the highest signals and this also correlates with the signal intensities from the IFA. The Western blot signals were given in mean intensities and were in accordance with the ELISA ODs ranging from 0.56 to 1.72 in triple experiments (mean values) as shown in Table 2. Intensity 1 indicates a weak positive signal (borderline), 2 indicates a strong signal and 3 a high signal compared to the positive control.
Table 2. Summary of positive results of hunters from western parts of Germany (group 1) and the Berlin area (group 2)
# Hunter group 1
Western blot (mean intensity)
Immunofluorescence (mean intensity)
Samples testing positive for tularemia antibodies.
Four of the five clearly Western blot and IFA positive-tested hunters were male; the mean age was 41 years. Three of them reported hunting and consuming hares and rabbits on a regular basis. All hunters lived in the bordering states of Hesse, Bavaria, Northrhine-Westfalia or Rhineland-Palatinate (Fig. 1, red boxes). Two hunters reported clinical symptoms: one hunter experienced severe pneumonia 2 years ago. At that time, tularemia was not considered as diagnosis. The second hunter suffered from chronic muscle pain and hepatitis of unknown origin. No clinical symptoms were reported from the remaining three hunters.
Figure 3 shows examples for a positive and a negative serum in the IFA from hunter 206 (group 1) and from hunter 571 from group 2, respectively. In (a) is shown the positive sample with a clearly visible ring structure around the bacteria (left picture), whereas it is absent in the negative probe (Fig. 3b, left). In the middle is the DAPI staining of the bacteria shown and the merge shows the overlap of Alexa®488 staining and DAPI.
From group 2 only three sera (3.3%) had suspicious ODs in ELISA (Table 2), and were tested in Western blot, but showed neither a lipopolysaccharide ladder in Western blot nor a significant ring structure in IFA. This means no hunter from this group was tested positive in any assay (95% CI 0–0.04%).
Comparison of the results from the previously performed cross-sectional study of German population [n=6883, randomly distributed over the whole of Germany (Porsch-Ozcurumez et al., 2004)] and group 1 hunters showed a clear elevated risk for hunters to be seropositive for F. tularensis (OR=7.7; P<0.001). The differences between hunter groups 1 and 2 were not significant.
Different methods are used to detect antibodies in sera. The commonly used agglutination test has the disadvantage of being time consuming and having problems with cross-reactivity, sensitivity and specificity (Schmitt et al., 2005). But the advantages are in terms of costs and the equipment needed, and is therefore preferable in laboratories without expensive instrumentation or during field investigations (Reintjes et al., 2002). However, cross-reactions with Brucella, Yersinia enterocolitica and Proteus spp. have to be considered (Porsch-Ozcurumez et al., 2004). The hunters were also screened for antibodies against Brucella and Yersinia, and showed negative results (data not shown).
The ELISA used in this study based on lipopolysaccharide recognition is suitable for high-throughput screenings when a large number of sera has to be investigated. To confirm the ELISA results, we used Western blot on SDS extracts of inactivated F. tularensis utilizing the typical lipopolysaccharide ladder for a reliable diagnosis of tularemia. This approach has been applied in former studies by Schmitt et al. (2005), revealing a sensitivity of nearly 100% and a specificity of 99.6% for the detection of Francisella-specific antibodies in the sera of exposed individuals.
High OD results in ELISA with negative Western blot findings may be caused by cross-reactivity, e.g. with contaminating heat shock proteins, which are resistant to proteinase K digestion and also appear weakly in Western blot. While in ELISA these cross-reactions with other components next to lipopolysaccharide cannot be differentiated depending on the assay type, in Western blot only the typical lipopolysaccharide ladder confirms a positive result. It was shown that the lipopolysaccharide fraction of F. tularensis induces highly specific antibodies; therefore cross-reactions did not occur with other bacteria (Schmitt et al., 2005).
The IFA used here verifies the results of the Western blot with comparable intensities of reactions, but unfortunately also with higher unspecific background. Therefore, the Western blot was preferred as a qualitative method compared to the IFA.
It is not known when the positive tested hunters became infected. Antibody titers are elevated even years after infection and might reside from prior exposure, and do not necessarily reflect recent or acute infections (Ericsson et al., 1994). Therefore, a serologic evidence of exposure is not tightly linked to an acute infection (Bevanger et al., 1994; Magnarelli et al., 2007) and could be a possible reason for weak intensities in Western blot. However, two hunters indicated tularemia-like diseases 2 years ago when the disease emerged in western Germany. The exact diagnosis could not be verified at that time.
The single person tested positive in our negative control group (0.4%) is consistent with former studies, where prevalences from 0.3% (Schmitt et al., 2005) and 0.2% (Porsch-Ozcurumez et al., 2004), respectively, were found in negative control groups. Because these studies were anonymized, it cannot be determined whether the seropositive individuals had contact with the Francisella bacterium. At present, the epizootic situation of tularemia in Germany is unknown (Al Dahouk et al., 2005).
Our study revealed that persons from a putative high-risk group for tularemia, i.e. hunters from western Germany with emerging tularemia, show a higher seroprevalence for the disease when compared with the entire population of Germany. A group of hunters from a region around Berlin, an area with less frequent disease, was seronegative. This could reflect a lower prevalence of the pathogen around Berlin, which coincides with the absence of reported cases of tularemia acquired from nature in this region. The recent outbreaks among hunters after a hare hunt in 2005 and the tularemia outbreak under groups of free-living monkeys confirm the presence of tularemia in animals in this region of western Germany. Although it is not clear how the tularemia pathogen circulates in Germany and how it can occasionally be transmitted to humans, our results indicate that there is an elevated risk of contracting the disease in some regions of Germany. Seroepidemiologic studies from Austria also revealed hunters as a major risk group for the prevalence of tularemia (Deutz et al., 2003).
To conclude, the detection of F. tularensis infections in hunters as a main risk group may be important to determine the epidemiological significance of the pathogen in selected sites or regions and provide geographic evidence of tularemia infections in nature. Overall, our results confirm the presence of tularemia in parts of Germany. Further work is required to characterize the real prevalence and epidemiology of this zoonosis more thoroughly in Germany.
The authors thank Alain Le Coustumier (Service de Biologie, Centre Hospitalier, Cahors, France) for providing the patients' serum samples, Dr Switkowski (AMD, Berlin, Germany) for the negative control sera and Derk Ehlert from the Senate Department of Urban Development, Berlin, for his kind support. The work was partially supported by a grant of the German Ministry of Health (1368-641).