Diagnosing <i>Francisella tularensis</i> infection in wildlife: improved diagnostics applied to emerging forms of tularemia
Diagnosing Francisella tularensis infection in wildlife: improved diagnostics applied to emerging forms of tularemia
Background. Francisella tularensis, the cause of tularemia, is a zoonotic pathogen with a wide animall host range. Emerging forms of tularemia, with new epidemiological presentation have been detected in wildlife in Europe in recent years. This project reviewed the diagnostic methodology at SVA, ANSES and DTU-VET, applying classical and new technologies to a selection of tularemia cases and described the pathology of emerging tularemia.
The aims of the studies were, i). to assess the suitability of the diagnostic methods in place at the three laboratories to detect and characterize emerging strains of F. tularensis and ii). to acquire knowldege (pathology and molecular typing) to contribute to the understanding of the epidemiology of emerging F. tularensis infection in wildlife hosts.
Results/value. The project revised, developed and incorporated state-of-the-art diagnostic methodology . It also allow technology transfer of the molecular diagnostic methods. An immunohistochemical (IHC) method was developed at SVA, which allowed demonstration of F. tularensis in relation to lesions. The pathology of tularemia in European brown hares (Lepus europaeus) was similar to that in mountain hares (Lepus timidus) and consisted in most cases of multifocal necroses in spleen, liver, lungs, bone marrow and other organs. The flourescent in situ hybridization (FISH) test done at DTU showed good agreement with the FAT test. At ANSES, 96.6% of tularemia cases were detected by multitarget real-time PCR(tul4, fopA, IsFtu2), 27/30 cases (90%) were detected by conventional nested PCR and 5/30 (16.6%) by culture on Francis medium. Molecular typing of was done at ANSES by MLVA on 87 F. tularensis isolates (60 from France, 19 from Sweden and 4 from Spain) and 4 reference strains. It distinguished 4 groups of F. tularensis strains, however, 95% of isolates were very similar and represented mainly groups 1 and 2. These results confirmed that F. tularensis subsp. holarctica isolates have low diversity and are highly similar whatever their origin in Europe. This study needs to be complemented using other tools such as canonical F. tularensis insertion-deletion element (Ftind) analysis and region differentiation analysis (RD).
In conclusion, a panel of diagnostic tests for tularemia including IIF (smears), IHC, FISH, PCR/SA, PCR/nested, Real time PCR, and culture showed variable diagnostic performance. Full agreement by all methods was obtained in 62.5% of the cases. The selection of diagnostic method(s) to be applied to tularemia should take into account the purpose of the testing and other factors, such as type of sample/material available, quality (autolysis), rapidity and costs.