Francisella tularensis is the etiological agent of the zoonotic disease tularemia and it is a facultative intracellular, highly infectious, Gram-negative bacterium (Sjöstedt, 2007). Diagnosis of tularemia may be challenging since it is a disease rarely seen by most physicians and may therefore not be immediately recognized, and, in addition, some patients present with non-specific symptoms (Anda et al., 2007; Petersen et al., 2017; Maurin, 2020). Tularemia cases are almost exclusively reported from regions in the Northern Hemisphere and although many countries report few cases, there are regions where the disease is endemic. The highest total number of tularemia cases have been reported from European countries, most notably, Sweden, Finland, Turkey, Hungary and Czech Republic, but in these countries, as well as in other countries with tularemia, there are very marked annual and seasonal variations and most outbreaks are local with random occurrence but with a predominance during late summer and autumn (Tärnvik et al., 1996; Tärnvik et al., 2004; Hestvik et al., 2015; Rossow et al., 2015).

Tularemia demonstrates distinct clinical presentations, dependent on the route of infection and varies from localized symptoms, such as skin lesions and lymphadenopathy, to severe respiratory disease and potentially life-threatening invasive disease (Tärnvik and Berglund, 2003; Anda et al., 2007; Maurin, 2020). The clinical presentation is also dependent on the bacterial subspecies and subspecies tularensis (type A), if inhaled, causes a disease with high mortality if untreated, whereas the other clinically important subspecies, holarctica, very rarely causes fatal disease, although individuals may become severely ill, in particular due to the respiratory form (Tärnvik and Berglund, 2003; Anda et al., 2007; Petersen et al., 2017; Maurin, 2020).

The predominant forms of tularemia are the ulceroglandular and respiratory forms. The former normally depends on spread of bacteria by vectors, predominantly mosquitoes or ticks, and the latter form on inhalation of contaminated dust (Hestvik et al., 2015; Abdellahoum et al., 2020; Telford and Goethert, 2020). Irrespective of form, tularemia results in effective protective immunity, and only a few cases of reinfection have been reported (Tärnvik and Berglund, 2003; Kirimanjeswara et al., 2008; Conlan, 2011; Elkins et al., 2016). The long-lived protection is dependent on cell-mediated immunity, as for many other intracellular bacterial infections. In fact, evidence indicates that vigorous cell-mediated responses are detected for at least 25 years in almost all individuals after tularemia or tularemia vaccination (Ericsson et al., 1994; Ericsson et al., 2001; Eneslätt et al., 2011). In the case of tularemia, also humoral immune responses, which are important for diagnosis, are unusually long-lasting, e.g., elevated IgM antibodies have been reported to be present 11 years after infection and low, but significant IgG antibody titers detectable in 50% of individuals up to 25 years after infection (Koskela and Salminen, 1985; Ericsson et al., 1994; Maurin, 2020).

The aim of the present study was to determine the kinetics of the IgM and IgG antibody titers up to one year after tularemia and to assess whether determination of individual titers is of diagnostic relevance.

Specific knowledge regarding the kinetics of humoral immune responses after infection is of much relevance from a clinical standpoint, since the information can be utilized to pinpoint when infection occurred and therefore be diagnostically important. Serological responses to many infections are characterized by IgM titers peaking within a couple of weeks and thereafter rather rapidly declining, sometimes disappearing within weeks or a few months, whereas IgG titers peak more slowly, but persist for much longer, at least many months (Vainionpää et al., 2015). Serological diagnosis of an acute infection is often based on the demonstration of high levels of IgM antibodies, or a significant increase in the levels of antibodies between two consecutive samples taken one to four weeks apart.

The use of serology for diagnosis of tularemia is well established and there are numerous studies demonstrating its utility, reviewed by Maurin (Maurin, 2020). A number of microagglutination tests (MAT) or ELISA tests have been evaluated and they generally demonstrate acceptable sensitivity of 85 - 95% and specificity of 90 - 95%. However, considering that tularemia is a rare disease in many countries, such specificities may still yield an unacceptable high false positive rate. An advantage of ELISA-based tests vs. MAT is that the former detects antibodies about a week earlier than the latter method (Maurin, 2020). The kinetics of the early humoral immune response after tularemia has been studied and, generally, few patients demonstrated significant antibody levels during the first week; 14% were found to be positive using ELISA, whereas 67% were positive during the second week, and 100% during the third and fourth weeks (Yanes et al., 2018). Also, other studies have observed that > 90% of the patients demonstrate significant IgG titers within 3-4 weeks (Carlsson et al., 1979; Koskela and Salminen, 1985; Syrjälä et al., 1986; Kilic et al., 2012). The results of the present study agree with the previous studies since we observed peak IgM levels one-month post infection and peak IgG antibodies two months post infection. Moreover, all of the patients seroconverted and demonstrated significantly elevated IgM and IgG titers. Notably, there are no consistent differences between the kinetics of the IgM and IgG antibody responses after tularemia, reviewed by Maurin (Maurin, 2020), in contrast to the serological responses to most other infections when IgG antibody responses remain elevated for much longer than do IgM antibody responses (Vainionpää et al., 2015).

It is well documented that cell-mediated and humoral immune responses after tularemia are very long-lived. In fact, cell-mediated immunity may be life-long since > 95% of individuals were found to present with significant cell-mediated immune responses 25 years after tularemia (Ericsson et al., 1994). At that time, humoral immunity had waned, but notably, 50% of the individuals still demonstrated IgG titers above cut-off, or threshold values (Ericsson et al., 1994). A Norwegian study demonstrated that 95% of individuals showed significant IgG titers 8 years after infection and a Finnish study that 100% showed significant IgM antibody levels 11 years after infection (Koskela and Salminen, 1985; Bevanger et al., 1994). Persistence of specific immune responses may be due to repeated exposure, however, in view of the low incidence of tularemia in the specific regions, this is an unlikely reason for the aforementioned long-lived immune persistence. The persistence of elevated IgG antibodies for decades is not unprecedented, however, since antibody responses after viral infections have been found to have half-lives of 50 years or more (Amanna et al., 2007). In support of the long-lived immune responses to F. tularensis, also in individuals vaccinated with the live vaccine strain of F. tularensis, cell-mediated immunity was found to persist for three decades without evidence of decline (Eneslätt et al., 2011).

The present study provides additional information regarding the kinetics after tularemia and demonstrated that there were elevated IgG and IgM antibody titers for at least 12 months after infection. Although the titers waned over time, compared to the average one-month IgG titer, a significant decrease for the group was not observed until the 12-month time point. Compared to the one-month IgM titer, there was significant decrease at the 4-month time point. Notably, the ≥ 69 year-group showed significantly lower antibody levels, a finding not reported previously. This is in agreement with the impaired B-cell responses occurring during ageing and concomitant increased susceptibility to various bacterial and viral infections among elderly (Frasca et al., 2011; Crooke et al., 2019; Palacios-Pedrero et al., 2021). A limitation of the study was the relatively few individuals included, however, the power was still sufficient to identify the age-related decline. Other, more subtle differences may be possible to identify if a larger cohort is analyzed.

Collectively, the data demonstrate long-term persistence of IgM and IgG antibody titers, both of which are significantly elevated during a period of 12 months. From a clinical perspective, this means that low-levels antibody titers are not indicative of acute or recent tularemia. Thus, serological diagnosis has to rely on the demonstration of significant increase of the antibody levels between two samples taken one to four weeks apart, concomitantly with a clinical presentation compatible with tularemia. Other authors have also made the same conclusions (Syrjälä et al., 1986; Bevanger et al., 1994; Yanes et al., 2018).

The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.

The studies involving human participants were reviewed and approved by the Regional Ethical Review Committee, Umeå 2016/335-31 and the Swedish Ethical Review Authority, 2019-01567. The patients/participants provided their written informed consent to participate in this study.

HL and KE performed the experiments. AS, JE, and HL designed the study. HL and AS analyzed the data and wrote the manuscript. HL, JE, KE, and AS reviewed the manuscript. All authors contributed to the article and approved the submitted version.